ENAMEL-DENTINE JUNCTION MORPHOLOGY OF

EXTANT HOMINOID AND FOSSIL HOMININ LOWER MOLARS

by Matthew Skinner

B.A. in Archaeology 2002, Simon Fraser University

A Dissertation submitted to

The Faculty of The Columbian College of Arts and Sciences of The George Washington University in

Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

May 18, 2008

Dissertation Directed By

Bernard A. Wood University Professor of Human Origins

ii

The Columbian College of Arts and Sciences of The George Washington University

certifies that Matthew Skinner has passed the Final Examination for the degree of Doctor

of Philosophy as of March 7, 2008. This is the final and approved form of the

dissertation.

ENAMEL-DENTINE JUNCTION MORPHOLOGY OF

EXTANT HOMINOID AND FOSSIL HOMININ LOWER MOLARS

Matthew Skinner Dissertation Research Committee:

Bernard A. Wood, University Professor of Human Origins, Dissertation

Director

Peter W. Lucas, Professor of Anthropology, Committee Member

Brian G. Richmond, Associate Professor of Anthropology, Committee

Member

iii

Dedication

This work is dedicated to all the fieldworkers whose efforts provided me with

museum and fossil specimens to include in this thesis, the many museum staff who curate

this material, and the past and present scientists who have studied teeth and provided me

with so much insight into these amazing biological structures.

“As the dentine, regarded from the morphological viewpoint, is the most integral

constituent of the tooth, its surface relief cannot be considered as a purely accidental

feature with no morphological significance” Franz Weidenreich, 1945

iv

Acknowledgments

I would like to begin by acknowledging Bernard Wood, the supervisor of my

doctoral studies in the Hominid Paleobiology Doctoral Program at The George

Washington University. First I would like to thank Bernard for accepting me as his

student and for encouraging me to get the most out of my time at GWU. Bernard was

very supportive of me both professionally and personally and provided me with many

valuable opportunities to participate in research projects, laboratory internships, and

fieldwork, all of which had a great influence on my education. “I am not interested in

what a person thinks, but what they can demonstrate” was a statement Bernard made to

me once, and which I have tried to remember during my own research endeavors.

Jean-Jacques Hublin, of the Max Planck Institute for Evolutionary Anthropology,

also supervised my thesis project. Without Jean-Jacques' support and guidance I could

not have conducted the projects included in this thesis. I am very grateful for the

opportunity that Jean-Jacques gave to me to conduct my thesis research within the scope

of his much broader research program on virtual paleoanthropology. I would like to thank

Jean-Jacques for sharing his enthusiasm for paleoanthropology with me and for the

critical and insightful observations he has contributed to this thesis.

I would like to thank Brian Richmond for his enthusiasm and the positive

approach that he brought to all aspects of the doctoral program at GWU. I benefitted

greatly from taking classes from Brian, from being his teaching assistant, and from his

guidance on a number of projects I conducted during my doctoral studies. Through his

research, teaching, and other scholarly activity Brian set a wonderful example to me and

the other students of the HOMPAL program.

v

I would like to thank Alison Brooks for instilling in me the importance of an

interdisciplinary approach to human evolutionary studies and of keeping up with all the

literature; which she seems to do so effortlessly. Also, for her support of my thesis

project and her encouragement to conduct my thesis research in Leipzig.

I would like to thank two people in particular for their assistance during my

doctoral studies. Tanya Smith first suggested to me the possibility to work in the

Department of Human Evolution at the MPI-EVA and this collaboration with my

department at GWU is responsible for the data upon which my thesis is based. Tanya also

encouraged me throughout my doctoral research, facilitated access to collections and

collaborators, and inspired me by her own scientific endeavors. Philipp Gunz provided

considerable assistance with the geometric morphometric analyses I employed in this

thesis. He kindly allowed me to use software routines that he developed, taught me how

to use them, and patiently helped with periodic difficulties I had with the analysis. Philipp

also provided valuable discussions regarding the interpretation of the results of many

analyses and in the broader implications of my research.

During my doctoral studies I was a student to many faculty from three academic

institutions. From GWU I would like to thank Peter Lucas, Patricia Hernandez, Robin

Bernstein, and James Clark. From The MPI-EVA I would like to thank Zeresenay

Alemseged, Katarina Harvati, Shannon McPherron, Tanya Smith, Philipp Gunz,

Kornelius Kupczik, Anthony Olejniczak, Luke Premo, Michael Richards, and Heike

Scherf. From the National Museum of Natural History I would like to thank Rick Potts

and Kay Behrensmeyer.

vi

I was fortunate to go through graduate school with students from two excellent

anthropology departments. I thank you all for your support, both personal and

professional, over the years and I look forward to being your colleague for many more.

From the Hominid Paleobiology Doctoral Program GWU: Janine Chalk, Paul

Constantino, Alexandra de Sousa, Tyler Faith, Felicia Gomez, David Green, Nicole

Griffin, Catherine Haradon, Amanda Henry, Nicolas Lonergan, Holly Mortensen, Lisa

Nevell, Robin Teague, Erin Marie Williams. From the Department of Human Evolution

at the MPI-EVA: Kate Britton, Kyungcheol Choy, Robin Feeney, Sarah Freidline, Maria

Hillier, Adeline Le Cabec, Olaf Nehlich, Anja Gumprich, Simon Neubauer, Philip Nigst,

Morgan Roussel, Nandini Singh, Sahra Talamo, Nicolas Zwyns and Friederike Kachel.

For their assistance with the administration of my doctoral studies I would like to

thank the administrative staff of the Department of Anthropology at GWU, including

Jonathan Higman, Gopy Mann, and Phillip Williams and the administrative staff of the

Department of Human Evolution at the MPI-EVA, including Diana Carstens, Silke

Streiber, and Cornelia Schicke.

For fruitful discussions during the course of my doctoral studies I acknowledge

Shara Bailey, Jukka Jernvall, Alistair Evans, Fred Grine, Mark F. Skinner, Paul

Constantino, Anthony Olejniczak, Kornelius Kupczik, Robin Feeney, Christine Verna,

Tanya Smith, Tim Weaver, Luke Premo, and Barth Wright.

I would like to acknowledge Heiko Temming, Andreas Winzer, Micha Gasch,

Dennis Reinhardt, Gert Wollney, Rasesh Kapadia for their assistance with technical

aspects of my thesis and for their participation in the computed tomography scanning

projects from which some of the thesis sample were derived.

vii

Access to dental specimens was kindly provided by the following individuals and

institutions: Mike Raath, Andre Keyser and Colin Menter of the University of

Witwatersrand, Johannesburg, South Africa; Francis Thackeray and Stephanie Potze of

the Transvaal Museum, Northern Flagship Institution, Pretoria, South Africa; Antonio

Rosas of the Museo Nacional de Ciencias Naturales, Madrid, Spain; Ottmar Kullmer of

the Senckenberg Museum, Frankfurt, Germany; Christophe Boesch, Mandy Jay and Mike

Richards of the Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany;

Rick Potts and David Hunt of the National Museum of Natural History, Washington, DC,

USA; Robert Asher, Hendrik Turni and Irene Mann of the Museum für Naturkunde,

Berlin, Germany; Emmanuel Gilissen and Wim Wendelen of the Royal Museum for

Central Africa, Tervuren, Belgium.

Various aspects of my thesis research received funding support from the

following: The George Washington University Selective Excellence Fellowship Program;

the National Science Foundation’s Integrative Graduate Education and Research

Traineeship Program; the European Virtual Anthropology Network; the Marie Curie

Research Training Network MRTN-CT-019564; the Spanish MEC Grant CGL-2006-

02131; The Max Planck Society.

Finally I would like to thank my mother for her support throughout my life and

for all of the sacrifices she made for her children. Also, for instilling in me a strong work

ethic and the drive to do a job well. I would like to thank my father for taking me to the

badlands of Alberta, Canada to look for dinosaur fossils as a small boy. I believe my

fascination with fossil teeth and my love of the outdoors derives from those hot days in

the prairies. I would also like to thank him for leading by his example as a scientist, for

viii

teaching me during my undergraduate degree at Simon Fraser University and for

encouraging me to apply to GWU where my doctoral studies began. I would like to thank

Jean McKendry for all of her support throughout my life and for her devotion to our

family. I would like to thank my brother Todd and his wife Andrea for all of their support

over the years and for allowing me to live vicariously through their lives in the ‘real

world.’ I would also like to thank my sister, Sarah Kate, for her support and for inspiring

me with her strength of character and love of life.

ix

Abstract of Dissertation

ENAMEL-DENTINE JUNCTION MORPHOLOGY OF

EXTANT HOMINOID AND FOSSIL HOMININ LOWER MOLARS

This thesis is comprised of four individual projects (chapters two to five) which

are based upon an examination of the enamel-dentine junction (EDJ) of lower molars in

various extant hominoids and fossil hominins. Collectively, these manuscripts represent

the first comprehensive analyses of EDJ morphology in a range of hominoid taxa and at

the high degree of resolution made possible through the use of micro-computed

tomography. They explore the taxonomic relevance of EDJ morphology, both

quantitatively and qualitatively, and in doing so reveal how detailed aspects of the outer

enamel surface morphology develop. They also provide insights into the developmental

processes that determine the form of the functional interface between the dentition and

the food, upon which natural selection acts.

The first chapter introduces the goals of the thesis and reviews relevant literature

that is pertinent to the various topics within the thesis. It also provides a detailed

discussion of the materials and methods used to image and analyze EDJ morphology.

Chapter two demonstrates that when the morphology of the EDJ can be captured in

sufficient detail, analysis of its morphology can discriminate between species and

subspecies of extant chimpanzees. Chapter three extends these findings to an analysis of

lower molars belonging to Australopithecus africanus and Paranthropus robustus from

southern Africa and demonstrates that EDJ morphology is distinctive both between each

taxon, as well as between first, second, and third molars of each taxon. Chapter four

examines the expression of four dental traits on the lower molars of a range of extant and

x

extinct hominoids (including fossil hominins). It demonstrates that these traits originate at

the EDJ, that the EDJ is primarily responsible for their degree of expression, and that

when examined across a wide range of taxa the morphological variability in the

expression of these traits can be considerable. The fifth chapter focuses specifically on

one dental trait, the protostylid, and examines its EDJ manifestation in samples of

Australopithecus africanus and Paranthropus robustus. The results of this analysis reveal

taxon-specific patterns in protostylid expression that are difficult to detect at the enamel

surface as well as evidence that current definitions of the trait itself should be re-

examined.

xi

Table of Contents

Dedication iii Acknowledgments iv Abstract ix Table of Contents xi Chapter 1. Introduction, Literature Review, Materials, and Methods 1 Chapter 2. Discrimination of Species and Subspecies of Pan Using the EDJ Morphology

of Lower Molars 48 Chapter 3. Enamel-Dentine Junction Morphology Distinguishes the Lower Molars of

Australopithecus africanus and Paranthropus robustus 80 Chapter 4. Dental Trait Expression at the Enamel-Dentine Junction of Lower Molars in

Extant and Fossil Hominoids 107 Chapter 5. Protostylid Expression at the Outer Enamel Surface and Enamel-Dentine

Junction of Lower Molars of Paranthropus robustus and Australopithecus

africanus 138

Chapter 6. Summary and Conclusions 161 References 167 Appendix A. Study Sample 180 Appendix B. Mathematica Code Used for Procrustes Superimposition 185

1

CHAPTER 1: INTRODUCTION, LITERATURE REVIEW, MATERIALS, AND

METHODS

Introduction

Teeth dominate the vertebrate fossil record due to their high degree of

mineralization and thus they are relied upon in the definition and diagnoses of fossil taxa.

Furthermore, because teeth do not remodel during the lifetime of an individual the

external morphology of the enamel and their internal microanatomy can be used to

reconstruct their development. Quantitative and qualitative analyses have demonstrated

that the external morphology of the enamel can readily distinguish genera, species and

even sub-species of hominoids, track patterns of modern human migrations and help

delineate regional grouping of modern humans (Johanson, 1974; Uchida, 1992, 1996,

1998a,b; Scott and Turner, 1997 and references therein; Pilbrow 2003, 2006a,b). The

morphology of the external enamel surface is also used to sort the hominin fossil record

into taxa and to reconstruct the evolutionary relationships among these taxa (e.g.,

Robinson, 1956; Sperber, 1974; Wood and Abbott, 1983; Wood et al., 1983; Suwa et al.,

1994; Bailey, 2002, 2006; Grine, 2004; Hlusko, 2004; Irish and Guatelli-Steinberg, 2003;

Guatelli-Steinberg and Irish, 2005; Bailey and Lynch, 2005; Bailey and Hublin, 2006).

The goals of this thesis are to extend this research through an investigation of whether

details of the shape of the interface between the enamel cap and the underlying dentine

(i.e., the enamel-dentine junction) can A) improve the utility of the morphology of the

crowns of lower molars of extant hominoids and fossil hominins for alpha taxonomy, and

B) improve our understanding of the development of morphological features (so-called

discrete dental traits) as seen on the external surface of lower molar crowns.

2

While teeth are relatively plentiful in the hominin fossil record and make up large

percentages of the known hypodigms for many hominin taxa they are variably worn.

Unerupted specimens can be pristine in their preservation of the original morphology of

the tooth crown, others have lost most of the morphology of the enamel cap, and heavily

worn specimens exhibit exposure of the underlying dentine. This variable preservation of

the original enamel surface limits our ability to compare those detailed aspects of crown

morphology that are necessary to discriminate between extant apes at the species and

sub-species level. With different levels of attrition the homology of many of the smaller

structures present on the crowns of fossil teeth is violated, and all that is left are relatively

gross measures of crown shape (e.g., length and width of the crown). Such gross

measurements are limited in their ability to discriminate fossil hominin taxa whose

discrimination, based on other skeletal morphology, is well supported. Thus, it is often

not possible to utilize the discriminatory potential of teeth in fossil samples. What is

needed is a structure that A) is homologous among teeth, B) carries information about the

detailed morphology of tooth shape, and C) retains that information even in teeth where

attrition has removed much of the external morphology of the enamel.

It has long been acknowledged that the enamel-dentine junction (EDJ), which

underlies the enamel cap of primate teeth, carries considerable information about the

shape of the external surface of the intact tooth crown (Kraus, 1952; Korenhof, 1960,

1961, 1982; Nager, 1960; Kraus and Jordan, 1965; Sakai et al., 1965, 1967a,b, 1969;

Sakai and Hanamura, 1971, 1973a,b; Corruccini, 1987a,b, 1998; Schwartz et al., 1998;

Sasaki and Kanazawa, 1999; Skinner et al., 2008) including taxonomically relevant shape

information (Corruccini, 1998; Olejniczak et al., 2004, 2006, 2007; Macchiarelli et al.,

3

2006; Suwa et al., 2007; Skinner et al, nd). Furthermore, the EDJ remains in a pristine

state throughout the initial stages of tooth wear (and even longer in thicker enameled,

relatively low-cusped hominin taxa). Study of the EDJ, however, has been limited by the

difficulty in imaging this surface non-destructively. This has prevented assessment of the

potential of this surface for determining the taxonomic affiliation of fossil specimens, for

understanding the development of morphological traits on the enamel surface, and for

characterizing variation in tooth crown shape in fossil hypodigms.

The two goals of this thesis are addressed through a series of questions related to

the morphology of the EDJ:

• Does the EDJ, like the unworn enamel surface, accurately

discriminate between hominoid species and subspecies? This question

is explored in chapter two through a comparison of lower molar EDJ

morphology in two species of Pan (Pan paniscus and Pan troglodytes)

and two subspecies of Pan troglodytes (verus and troglodytes)

• Can the EDJ, preserved in partially worn fossil hominin teeth, be used

to assess the taxonomy of fossil specimens? This question is the topic of

chapter three which compares lower molar EDJ morphology between two

species of hominin from southern Africa: Australopithecus africanus and

Paranthropus (Australopithecus) robustus.

• What is the contribution of the EDJ to the presence and

morphological expression of discrete dental traits on lower molars?

This question is addressed in chapter four through an examination of the

EDJ expression of four lower molar dental traits (cusp 6, cusp 7,

4

protostylid and trigonid crest pattern) in a range of extant and extinct

hominoid taxa (including fossil hominins).

• Are there morphological differences in protostylid expression at the

EDJ between Au. africanus and P. robustus? This question is addressed

in chapter five through an analysis of protostylid expression at the enamel

surface and EDJ of a large sample of molars.

The data used to address each of these questions are digital reconstructions of the

surface of the enamel cap and the EDJ of molar teeth acquired non-destructively through

microcomputed tomography (µCT). This technology allows the production of high

resolution reconstructions of each surface that are necessary to examine the detailed

morphological features present on molar teeth. These surface reconstructions are

analyzed qualitatively, through visual inspection, and quantitatively, through traditional

mensuration as well as within a geometric morphometric framework. The latter allow

both a quantitative analysis of complex shapes and a means of visualizing the shape

differences present between both taxa and tooth type (e.g., first, second, and third

molars).

Chapters two to five are written in the form of individual manuscripts. This is

because some manuscripts are already published (Chapter 4) and others are in various

stages of submission at the time of the writing of this thesis. This first chapter presents a

review of the literature relevant to the topics addressed in each chapter and a detailed

discussion of the materials and methods incorporated into the thesis. The final chapter of

the thesis summarizes the results of the previous chapters, and discusses the implications

5

of the results for our understanding of how tooth morphology develops and how variation

in tooth morphology is incorporated into dental and paleoanthropological research.

Terminology

There are a number of terms used thoughout this thesis that require some

explanation. The first set relates to the use of the taxonomic terms hominid, hominine and

hominin and, in recognition of the genetic similarities between Pan and Homo sapiens,

follows Wood and Richmond (2000). The term hominid refers to members of the family

Hominidae including all the extant great apes (i.e., Gorilla, Pan, Pongo, and modern

humans), all fossil apes more closely related to these extant taxa than to any other extant

taxon, and fossil hominins – see below), the term hominine refers to members of the

subfamily Homininae (including chimpanzees, bonobos, modern humans, fossil panins

and fossil hominins – see below), the term panin refers to members of the tribe Panini

(i.e., modern chimpanzees and bonobos and all fossil taxa more closely related to modern

chimpanzees and bonobos than to any other extant taxon), and the term hominin refers to

members of the tribe Hominini (i.e., modern humans and all fossil taxa more closely

related to modern humans than to any other extant taxon).

The nomenclature used to identify the cusps on lower molars is that commonly

used in vertebrate paleontology (Fig. 1.1). This includes the protoconid (mesiobuccal

cusp), metaconid (mesiolingual cusp), entoconid (distolingual cusp), hypoconid

(distobuccal cusp), and hypoconulid (distal cusp). Underlying each of these cusps is a

dentine horn referred to by the same name (e.g., protoconid dentine horn). The terms Ma

and Ka stand for millions of years ago and thousands of years ago, respectively.

6

History of EDJ imaging

Many attempts to image the EDJ have incorporated the chemical removal the

enamel cap (e.g., Kraus, 1952; Nager, 1960; Sakai et al., 1965, 1967a,b, 1969; Sakai and

Hanamura, 1971, 1973a,b; Corruccini, 1987b, 1998; Corruccini and Holt, 1989; Sasaki

and Kanazawa, 1999; Kono et al., 2002). This method provided reasonable access to EDJ

morphology, but it required prior preservation of the enamel surface (usually by casting)

and suffered from potential damage to the EDJ during the removal of adhering enamel

(e.g., see Corruccini and Holt, 1989). A fortunate opportunity to examine EDJ

morphology in molar teeth occurred with the discovery by G. H. R. von Koenigswald of

many hundreds of naturally preserved enamel caps from around Sangiran, Central Java,

Indonesia. These modern human teeth derive from medieval burials and were collected

over a five year period from 1935 to 1940 (Weidenreich, 1945, Korenhof, 1960). Plaster

endocasts of these enamel caps formed the basis of a series of publications on the

collection (Korenhof, 1960, 1961, 1978, 1982). This series of teeth and endocasts

revealed considerable information about the degree of concordance between the EDJ and

enamel surface (see below) but the methodology could not reliably document the

presence or absence of fine details of the morphology of the EDJ. For example, Korenhof

(1960) noted a lack of any definitive features underlying mesial marginal ridge tubercles

on the upper molars, however, corresponding EDJ features have been noted subsequently

by other authors (Kraus and Jordan, 1965).

Another major source of information about EDJ shape came from developing

tooth germs removed from jaws. One of the most comprehensive studies of this kind was

7

an analysis of deciduous and permanent molars undertaken by Kraus and Jordan (1965),

which included illustrations of the growth of the EDJ and the subsequent pattern of

enamel deposition over its surface. Photographs of the tooth germs, chemically-stained to

reveal the mineralized portions, demonstrated that morphological features visible at the

enamel surface are present on the EDJ (more precisely the inner enamel epithelium of a

developing tooth germ) prior to enamel deposition. Another important study using this

method was that published by Kraus and Oka (1967) in which they demonstrated that

“wrinkled” enamel on the surface of hominoid and modern human teeth derived from

ridges and crests present on the EDJ prior to enamel deposition. In the most recent and

comprehensive analysis of primate tooth germs, Swails (1993) examined monkeys, apes

and modern humans to compare taxonomic and phylogenetic signals in the ontogenetic

trajectory of molar tooth germs.

Computed tomography (CT) is an ideal methodology for studying internal

structures because it is non-destructive to the tissues and provides 3D volumetric data.

CT has been employed to image internal tooth structure, but many of these studies were

concerned with imaging the EDJ in order to measure enamel thickness rather than to

assess EDJ morphology itself (Conroy, 1991; Grine, 1991, Macho and Thackeray, 1992;

Conroy et al., 1995; Schwartz et al., 1998). The study by Schwartz and colleagues (1998)

examined the concordance between the EDJ and OES with regard to Carabelli’s trait

expression in hominin upper molars. Their systematic attempt to address the contribution

of the EDJ to this dental trait provided equivocal support for the consistent presence of

the trait at the EDJ. This was because of limited spatial resolution and the limitation that

this feature could only be assessed within a single slice.

8

The spatial resolution of medical grade CT (e.g., 0.5 – 1.0 mm per pixel) is not

sufficient to accurately assess detailed morphological features on the EDJ, or their

correspondence with equivalent features on the outer enamel surface (OES). In the last

decade so-called micro-computed tomographic (µCT) scanners have become available

for anthropological research. These tomographic scanners have a superior spatial

resolution reaching approximately 5-50 micrometers (µm) for most dental

anthropological applications, allowing surface reconstructions of the EDJ and OES to be

generated that are able to capture the complex morphology present on tooth crowns. The

majority of studies utilizing µCT have focused primarily on enamel thickness with less

discussion of EDJ shape variation (Kono et al., 2000; Kono, 2004; Suwa and Kono, 2005;

Olejniczak, 2006; Olejniczak and Grine, 2006; Smith et al., 2006b) although more recent

publications have used µCT to address the taxonomic distinctiveness of enamel thickness

and EDJ shape in extant and fossil taxa (Olejniczak et al., 2004; Olejniczak and Grine,

2005; Olejniczak, 2006; Macchiarelli et al., 2006; Kunimatsu et al., 2007; Suwa et al.,

2007; Olejniczak et al., 2008, nd).

Two-dimensional studies of EDJ shape

One method of studying EDJ shape is to examine it in cross-section. One reason

for this is practical as the production of histological sections used for study of enamel

thickness gives access to EDJ shape as well. As large histological collections of modern

human teeth (and a small sample of primates and fossil hominins) have already been

produced, it is a good opportunity to further utilize these specimens. Olejniczak and

colleagues (2004) examined EDJ shape in the maxillary molars of anthropoid primates

9

and concluded that EDJ cross-sectional shape through the mesial cusps discriminates

between taxa at the family, genus and species level. An analysis of modern human molars

by Smith and colleagues (2006a) found some evidence for population differences and

metameric variation along the molar row. Olejniczak and colleagues (2008) examined

dentine horn height in 2D (based on single µCT slices) in Gigantopithecus blacki and

noted similarities with extant Pongo in having relatively short dentine horns compared to

African apes and humans. An analysis of dentine height, assessed from radiographs,

showed no significant differences between modern humans and Neanderthals although

linear measurements incorporating enamel height, dentine height and pulp accurately

discriminated between the two taxa (Zilberman et al., 1992). Shimizu (2002) examined

cross-sectional images of cercopithecoid monkeys and suggested that the shape of the

EDJ and the distribution of enamel over the dentine horns contributed to the maintenance

of functional efficiency as the molars worn down. This is one of the few studies that

directly addresses the role that tooth crown conformation plays in the functional

morphology of teeth. In summary, 2D studies of EDJ shape have shown promise for

addressing taxonomic and functional questions but have been limited in their taxonomic

scope and because they assess a single section through a complex 3D structure are

potentially misleading with regard to the assessment of some morphological features.

Three-dimensional studies of EDJ shape

In Butler’s (1956) comprehensive review of the current knowledge of how molars

grow were the following observations relevant to the present study: a) minor features of

the OES may or may not involve the EDJ, b) wrinkling on the OES of hominoids has an

10

EDJ component, c) enamel deposition can mask the presence of small cuspules

originating at the EDJ, d) that the EDJ and OES will be more similar in shape in thin-

enameled than in thick-enameled taxa, e) cusps can be defined as the locations where the

formation of dentine and enamel begins, and f) ridges are less developmentally stable

than cusps.

Kraus (1952) examined the correlation between bi-apical cusp diameters and

angles in modern humans to determine the reliability with which cusp configurations

could be reconstructed in worn fossil teeth. His results did not support a strong predictive

relationship between EDJ and OES morphology for such variables. Kraus and Jordan’s

(1965) analysis of deciduous and permanent molar tooth germs identified the basic

sequence by which each molar attains its overall shape during soft tissue growth and

mineralization. They noted variability in the sequence of cusp coalescence during

mineralization likely linked to variation in EDJ shape, and they noted many similarities

between modern human molar EDJ morphology at various stages of development and the

molar morphology of extant and extinct primates, mammals and reptiles. Their

observations suggested to them that the processes by which teeth form different shapes

have been conserved across many lineages and over deep evolutionary time and that the

developmental stages through which modern human primary molars pass “recapitulate

the adult forms of fossils which are postulated as representatives of stages in the

evolution of the human molars (Kraus and Jordan, 1965:164).”

As mentioned above the series of papers by Korenhof (1960, 1961, 1978, 1982)

represent one of the most comprehensive analyses of modern human molar EDJ

morphology. Among his many observations were that the EDJ a) preserves more

11

phylogenetically conservative morphology than the OES, b) shows details that are not

evident at the enamel surface, and c) is indicative of how features present on more

generalized primate teeth have been incorporated into structures on modern human teeth.

Korenhof also proposed theories about evolutionary trends in particular aspects of molar

morphology that are present at the EDJ and can be compared to molar features of extant

and fossil primates.

Sakai and colleagues published a series of papers on the EDJ and OES of all tooth

types of modern humans (Sakai et al., 1965, 1967a,b, 1969; Sakai and Hanamura, 1971,

1973a,b). The close similarity in shape between the EDJ and OES of major crown

features is evident in their publications indicating the origin of these features on the EDJ.

With regard to the phylogenetic signal preserved in each surface they conclude that “the

characters of the dentinoenamel junction show phylogenetically more primitive or

conservative conditions in relation to the exterior surface of the enamel layer…” and that

“a morphological study of the dentionoenamel junction should play an important role in

morphologic interpretation of various dental traits and studies on phylogeny of the

dentition (Sakai and Hanamura, 1973b).”

Corruccini and colleagues published a series of papers examining the EDJ of

primates (Corruccini, 1982, 1987a,b, 1998, Corruccini and Holt, 1989). Corruccini

(1982) confirmed the clarity with which morphological features, such as accessory cusps

and crests, can be seen at the EDJ and noted similarities between modern human and

chimpanzee molar EDJ morphology that are less evident at the enamel surface. In a

taxonomically broad study of primate EDJ molar morphology Corruccini (1987a) noted a

number of cases of features present at the EDJ that were not seen at the OES.

12

Interestingly, cases of EDJ hypocones with no OES equivalent, as well as, the opposite

pattern were present in modern human molars. Based on EDJ morphology it was also

suggested that the hypocone may not be developmentally homologous among living

primates, having evolved from different portions of the crown in different taxa

(Corruccini, 1987a, Corruccini and Holt, 1989). Corruccini (1998) found that the EDJ

tended to be more similar in shape to the expected plesiomorphous condition and showed

similarities between modern humans and African apes and between callitrichines and

pitheciines that are not evident at the OES.

Surface reconstructions of the EDJ and OES, derived using µCT, have also been

used to establish and compare developmental trajectories within and between modern

human teeth (Smith et al., 1997; Avishai et al., 2004). They noted that the increase in

intercusp distance at the OES was due to a difference in cusp angulation rather than an

increase in enamel thickness and that EDJ morphology and enamel deposition can

contribute in different ways to total cusp size and volume between cusps on the same

molar crown.

Sasaki and Kanazawa (1999) examined morphological traits, including the

protostylid and trigonid crest pattern, on the EDJ of lower deciduous molars of modern

humans. Echoing the findings of earlier studies it was shown that the EDJ preserves a

more accurate record of the presence of these features and that small features at the EDJ

can be masked by thick enamel. Suwa and Kono (2005) noted the consistent reduction in

dentine horn height between first and second lower molars in modern humans.

Macchiarelli and colleagues (2006) commented on the apparent EDJ surface complexity

of Neanderthal molars compared to modern humans, but due to the very small sample

13

size in their analysis and the simplistic quantification of surface complexity (i.e.,

measured as EDJ surface area) this apparent distinction requires further study.

Due to small sample sizes in the majority of these studies, emphasis was placed

on the degree to which the EDJ preserved plesiomorphic morphology and how closely

EDJ morphology tracked inferred phylogenetic relationships among taxa, rather than on

the reliability of the EDJ to discriminate among closely related taxa (e.g., at the species or

subspecies level). One of the goals of this thesis is to compare larger samples of relatively

few taxa to assess variability in dental trait morphology and the degree to which the EDJ

can reliably discriminate closely related taxa.

Odontogenesis

This section outlines current knowledge of how tooth crown morphology

develops and highlights aspects of development that can lead to morphological variability

of crown features. As this thesis focuses on identifying and interpreting molar crown

variability in extant and fossil hominids, this review serves as the basis from which

variation in EDJ morphology, preserved in fully formed molars, can be interpreted. It

begins with a review of modern human tooth development as understood through the

histological analysis of tooth germs following Ten Cate (2007). This is followed by a

review of what is known about the developmental genetics of mouse tooth growth as this

field is providing significant advances in our understanding of how teeth grow. Finally, I

discuss the implications of this new developmental genetic paradigm for interpreting

morphological variation of molar tooth crowns in primates and fossil hominins.

14

Embryology of odontogenesis based on histology. Modern human teeth are

initiated from the maxillary, frontonasal and mandibular processes of the developing

embryo and their formation is divided into five stages: primary epithelial band (dental

lamina), bud, cap, bell, and crown stages. In the first stage horseshoe-shaped primary

epithelial thickenings form in positions corresponding to the future dental arches in the

upper and lower jaws. Continued epithelial cell proliferation, within the dental lamina,

results in localized epithelial thickenings that mark the locations of future teeth. The bud

stage begins when the oral epithelium invaginates into the underlying mesenchyme

resulting in the formation of a bud-like structure.

The early cap stage is characterized by a condensation of mesenchymal cells

beneath the epithelial ingrowth that is caused by the failure of these cells to produce

extracellular matrix. The late cap stage is characterized by the formation of the dental

organ (containing epithelial cells), which sits over the dental papilla (containing

condensed mesenchymal cells) like a cap. The dental follicle, also comprised of

condensed mesenchymal cells, surrounds the dental papilla and dental organ. Collectively

these three structures form the tooth germ.

The bell stage of tooth development is characterized by the histodifferentiation

and morphodifferentiation of epithelial cells within the dental organ. These cells

differentiate into the outer enamel epithelium (at the outside surface of the dental organ),

the inner enamel epithelium (over the dental papilla), the stellate reticulum (within the

dental organ and surrounded by the outer and inner enamel epithelium), and the stratum

intermedium (located between the inner enamel epithelium and the stellate reticulum).

The stellate reticulum contains glycoaminoglycans which are hydrophilic and provide for

15

a high proportion of water within the dental organ to maintain its shape and separation

between the inner and outer enamel epithelia.

The crown stage is characterized by continued mineralization of the dental hard

tissues that commenced at the bell/crown stage boundary. Beginning at the site of future

cusp tips and moving down the cusp walls, the cells of the inner enamel epithelium cease

mitotic activity and elongate into column-shaped cells with the nucleus adjacent to the

stratum intermedium. Subsequently, the cells of the dental papilla beneath the epithelium

increase in size and differentiate into odontoblasts. After the odontoblasts deposit

predentine and dentine begins to form, the columnar cells of the inner enamel epithelium

differentiate further into fully formed ameloblasts, which begin to secrete enamel matrix

on top of the dentinal surface. As new odontoblasts continue to differentiate, moving

down the inner surfaces of the cusp walls, new ameloblasts also differentiate cervically,

while more mature ameloblasts begin to deposit enamel in long prisms as they migrate

towards the eventual crown surface. The cervix of the tooth is located at the cemento-

enamel junction where ameloblast differentiation and enamel formation ceases.

Odontoblasts continue to proliferate apically to form the tooth roots. Cementum,

deposited by cementoblasts, covers the root surface and anchors the tooth into the

surrounding alveolar bone by incorporating fibers of the periodontal ligament into its

sequentially deposited hard tissue layers.

The dentition of modern humans consists of both deciduous and permanent sets of

teeth. The morphogenesis of the incisors, canines and premolars of the permanent

dentition progresses through the same stages of development as their deciduous

precursors, beginning with the formation of a diverticulum on each developing deciduous

16

tooth germ that will eventually form the permanent tooth bud. The molars of the

permanent dentition, which in modern humans have no deciduous predecessors, develop

from posterior migrations of the dental laminae beneath the lining epithelium of the oral

mucosa and into the underlying mesenchyme, as the mandible and maxilla increase in

size.

Developmental genetics of murine odontogenesis. Recent experimentation using

mice has identified many of the primary genes and signaling pathways responsible for the

formation of the dental tissues and their temporospatial activity during tooth growth. This

work has been the impetus for the generation and testing of new developmental models

that attempt to explain the processes behind the dental phenotypes that are present in

mice and other mammal groups.

The development of all mammalian teeth involves reciprocal signaling between

the oral epithelium and the neural crest-derived mesenchyme and is thus limited to the

first branchial arch as this is the only location in which these two tissues occur. While

teeth can be initiated in neural crest-derived mesenchyme from the second branchial arch,

no initiation occurs in the presence of non-oral epithelium (Lumsden, 1988; Mina and

Kollar, 1987). The pervasiveness of cranial neural crest (CNC) cells in the development

of the teeth and the mandible in mice has been demonstrated by Chai and colleagues

(2000) who determined that CNCs contribute to the development of the condensed dental

mesenchyme, dental papilla, odontoblasts, dentine matrix, pulp, cementum, periodontal

ligaments, the mandible and the articulating disc of the temporomandibular joint.

Tissue recombination experiments, in situ hybridization, and gene knockout

experiments have identified the numerous genes and transcription factors involved in the

17

various stages of tooth morphogenesis. Members of four families of signaling proteins

are iteratively and reciprocally expressed between the oral epithelium and mesenchyme

throughout murine tooth morphogenesis: bone morphogenetic proteins (BMPs),

fibroblast growth factors (FGFs), Sonic hedgehog (Shh) and Wnt proteins (Ferguson et

al., 2000a). Also, the same transcription factors are often present at each stage of tooth

development (e.g., p21, Msx2, Lef1, Barx1, and Dlx2).

A pattern of nested expression of FGF8 and BMP2 and BMP4 is believed to

control the spatial pattern of Pax9 expression associated with tooth bud formation

(Neubuser et al., 1997). Shh has also been linked to bud formation as it is expressed in the

tip of the epithelium that invaginates into the mesenchyme (Dassule et al., 2000). Other

signaling molecules linked to bud formation by knockout experiments include Msx1 (all

teeth arrested at the bud stage) (Satokata and Maas, 1994), Lef1 (failure of dental papilla

formation resulting in rudimentary bud formation) (van Genderen et al., 1994), Gli2-/-

Gli3+/- (no maxillary incisor buds), Gli2-/-Gli3-/- (all teeth arrested at the bud stage),

Dlx1-/-Dlx2-/- (no maxillary molar buds), and Activin A-/- (no incisor or mandibular

molar buds) (Line, 2003). Redundancy is evident in many of these signaling molecules,

as only in double knockout mutants is any phenotypic effect evident.

The shape of the tooth crown is determined during the cap and bell stages.

Specifically, this involves the differential proliferation of epithelial and mesenchymal

cells controlled in time and space by a structure called the enamel knot. This differential

growth results in the folding of the epithelium that creates the basic shape of the crown,

with the shape of the occlusal surface dictated by the subsequent growth of the cusp(s).

The enamel knot is a non-proliferative signaling center similar to the apical ectodermal

18

ridge (AER) present in developing limbs, and its induction is a prerequisite for the tooth

to develop into the cap stage (Jernvall and Thesleff, 2000). The non-proliferation of

enamel knot cells is thought to be maintained by the high prevalence of FGF receptors in

the enamel knot area and the expression of p21, a cyclin-dependent kinase inhibitor that

is associated with cells ceasing to divide and with beginning terminal differentiation (p21

is also expressed in the non-proliferating AER in limbs). Given that knockouts of p21 do

not result in altered adult dental phenotypes it is likely that p21 is functionally redundant

with other inhibitors around the enamel knot (Jernvall and Jung, 2000). By the late cap

stage the enamel knot has disappeared through apoptosis in association with BMP4

expression (Jernvall et al., 1998).

Developmental data contradict arguments that complex, multicuspid teeth evolved

through the amalgamation of numerous monocuspid teeth (Donoghue, 2002). In

multicuspid teeth, the apoptotic disappearance of the primary enamel knot, is followed by

the formation of secondary enamel knots at the sites of future cusp tips. Like primary

enamel knots, secondary enamel knots are non-proliferative and are removed

apoptotically in association with BMP4 expression (Jernvall and Thesleff, 2000). The

spacing of secondary enamel knots (and their associated cusps) is important in

determining crown shape. It is thought the nested expression of FGF4 (linked to epithelial

cell proliferation and cusp formation) and BMP4 and/or Shh (inhibiting the Fgf4 signal)

regulate the distance between cusps and overall cusp size (Jernvall and Thesleff, 2000).

FGF4, Shh, Lef1 and p21 show similar correlations between pre-cusp expression patterns

and eventual cusp morphology, suggesting that they are all involved in cusp patterning

19

(Jernvall et al., 2000). Edar has also been implicated in the determination of cusp number

and tooth number in mice (Tucker et al., 2004).

In summary, odontogenesis occurs through sequential and reciprocal signaling

within the oral epithelium, and between the oral epithelium and the underlying

mesenchyme, and it involves the same signaling families (e.g., FGFs, BMPs, Wnts and

Shh) and the iterative use of the same signaling pathways, which are controlled, in large

part, by primary and secondary enamel knots (non-proliferative signaling centers similar

to the AER of developing limbs). The complete dental arcade of modern humans then can

been viewed as the collective development of individual teeth that have either single

secondary enamel knots in the case of monocuspid teeth, or multiple secondary enamel

knots in the case of multicuspid teeth. Based on this research, new models have been

proposed account for both the different tooth types in heterodont dentitions and cusp

patterning on the crowns of multicuspid teeth.

Determination of tooth type: the odontogenic homeobox code model. Butler

(1956) proposed a gradient model of dental patterning in which concentration gradients

of unspecified molecules determine separate fields in which incisors and molars develop.

Osborn (1978) suggested a clone model in which different populations of mesenchymal

cells populated the first branchial arch which eventually gave rise to incisor and molar

specific mesenchymal cells. Based on recent developmental genetic research, the

odontogenic homeobox code (OHC) model posits that teeth are serially homologous

structures and tooth type is determined by nested and restricted homeobox gene

expression in the maxillary and mandibular mesenchyme (Sharpe, 1995; Thomas et al.,

1997). It is currently believed that it is the coexpression of homeobox genes in the

20

mesenchyme, induced by signals from the oral epithelium, which determines tooth type

in mice. Molars grow where Barx1 and Dlx2 overlap, and incisors grow where Msx1 and

Msx2 overlap (Ferguson et al., 2000a; Ferguson et al., 2000b).

This model was derived in part from gene knockout experiments that resulted in

either the loss of different tooth classes (e.g. maxillary molars) or a change in tooth shape

(e.g. molars forming in the incisor region). For example, using exogenous Noggin protein

beads to inhibit BMP4, which normally restricts expression of Barx1 to the presumptive

molar region of the mandible, Tucker and colleagues (1998) were able to induce

expression of Barx1 in the presumptive incisor region of the mandible, and transform

tooth identity from an incisor to a molar. It seems reasonable that the determination of

tooth types present in primates that are not present in mice (i.e., premolars and canines) is

similarly controlled by the nested expression of homeobox genes. Under the OHC model

there is not one specific gene that is responsible for each tooth type, and because the code

is overlapping it can specify a wide range of subtle differences in tooth shape (Cobourne

and Sharpe, 2003).

Patterning cascade mode of cusp development. A patterning cascade mode of

tooth development, inspired by the developmental genetic research discussed above, has

recently been proposed to explain the variation in cusp size, cusp spacing and total cusp

number observed in multicuspid tooth crowns (Weiss et al., 1998; Polly, 1998; Jernvall,

2000; Jernvall and Thesleff, 2000). The primary tenet of this model is that the

mechanisms inducing cusp formation and mechanisms controlling growth of cusps are

both active throughout crown development; this continuous interaction between inductive

and morphogenetic mechanisms is referred to as being morphodynamic. Under a

21

morphodynamic model cusp initiation is sequential and the location and size of later

forming cusps is influenced by the location and size of previously forming cusps

(Salazar-Ciudad et al., 2003). In developmental terms, the positioning of later forming

secondary enamel knots (and their associated cusps) is dependent on the positioning of

previously forming secondary enamel knots. For example, when a broader inhibition field

around the initial enamel knot(s) increases cusp spacing, cusp number will either be

reduced or the later-forming cusps will be smaller in size. Conversely, when a narrower

inhibition field decreases cusp spacing the number of cusps will be increased or the later-

forming cusps will be larger.

This prediction has been experimentally demonstrated by a number of studies.

Kangas and colleagues (2004) determined that altering of expression levels of a signal

gene (Eda) had profound effects on the resulting dental phenotype with underexpression

resulting in smaller molars with fewer cusps, and overexpression resulting in the

formation of supernumerary molars and molars with more cusps. Kassai and colleagues

(2005) identified ectodin, a bone morphogenetic protein inhibitor, as one contributing

factor controlling the size and spacing of mouse enamel knots, as well as, the number of

molars.

Jernvall (2000) stresses that while large phenotypic differences can result from

minor developmental changes affecting cusp spacing and growth, it is not always

necessary to have a change in developmental information for cusp patterning to evolve

(in terms of either the gain or loss of cusps). For example, if other factors are held

constant under the patterning cascade model, an increase in overall tooth crown size will

tend to result in the appearance of more cusps and a decrease in crown size will tend to

22

diminish cusp number. Cai and colleagues (2007) in a recent analysis using

heterospecific recombinations of tissues of molar tooth germs of mice and rats

determined that dental epithelium was responsible for cusp size and dental mesenchyme

responsible for tooth size. Thus, cusp number and patterning is regulated by overall tooth

size and cusp size. This could be an important factor when weighing the phylogenetic

significance of variation in cusp number between closely related taxa of differing body

size. Jernvall and Jung (2000) note that teeth with complex, multicuspid crowns may

have independent patterning cascades for different parts of the tooth (e.g., trigonid vs.

talonid) or different groups of cusps (e.g., paracone-protocone-Carabelli cascade and a

paracone-metacone cascade). If true, such a developmental scenario would have clear

implications for interpreting patterns of variation and covariation of cusp morphology

across the tooth crown.

Interpreting molar crown variation within a developmental framework. What are

the implications of the development of molar teeth, as discussed above, for the

interpretation of morphological variation in the EDJ. Under a simple model that is

applicable to the study of fully formed teeth, two processes occur that are primarily

responsible for the morphology of the tooth crown that erupts into the mouth. The first

process is the growth and folding of the inner enamel epithelium (IEE) prior to

mineralization. It is within the IEE that enamel knots form and their location marks the

site of future dentine horns (pointed, conical structures similar in shape to, and

underlying the cusps seen on the OES). Dentine horns appear sequentially on the

developing IEE. The timing of initiation and spatial relationships between these dentine

23

horns dictate the overall appearance of the tooth crown in terms of its cusps. The final

form of the IEE is preserved in the tooth crown as the EDJ.

The second process involves the mineralization of the tooth germ. Deposition of

enamel matrix on the tooth germ begins at the tips of the dentine horns and proceeds

down their slopes. These individual islands of mineralizing enamel eventually coalesce in

the valleys between dentine horns and the mineralization front continues extending until

it reaches the cervix, or neck, of the tooth. Because the IEE can continue to grow between

mineralizing dentine horns until they coalesce, the early process of IEE growth and

folding and the later process of enamel deposition are not completely distinct and the

former is likely partially influenced by the latter. How the enamel deposition alters the

shape of the outer enamel surface, from a default shape that is the EDJ, is dictated by the

rate at which ameloblasts differentiate, their daily secretion rate and the duration over

which they secrete enamel. It is important to remember that both the EDJ and enamel

distribution can express variation in a population, upon which natural selection can act.

Thus, changes in tooth crown morphology may involve early and/or late developmental

processes.

“Only by knowing the developmental processes generating a character can we

make reliable inferences on inheritance and character independence” (Jernvall and Jung,

2000:185). This statement reflects the increasing role that developmental genetics will

play as researchers attempt to evaluate the phylogenetic signal present in different aspects

of tooth crown morphology. Morphological characters used in phylogenetic systematics

are assumed to be independent of each other and to satisfy the criteria of homology.

Understanding their development and evolvability is an important step in validating these

24

assumptions. Developmental research is also offering new definitions of homology with

which systematists must grapple: “homology is not just a static genetic code readable

deep inside the genome, but rather, it is a readout of the information stored in the

dynamic cusp-making program (Jernvall and Jung, 2000:187).”

In an attempt to evaluate dental characters from a developmental perspective

Jernvall and Jung (2000) grouped dental characters into four categories: initiation,

termination, cusp shape and configuration characters. Variation in the timing of initiation

of cusps affects individual cusp size and cusp number. Small cusps are more likely to

show size differences and there would be higher potential for homoplasy in the

presence/absence of small cusps. Specific examples outlined by Jernvall and Jung (2000)

are hypocone size, nanopithexfold size, paraconule size, talonid height and cristid obliqua

size. Variation in the timing of the termination of growth affects the morphology of both

individual cusps and the tooth crown as a whole. Early termination of crown growth

results in shorter crowns and a decreased frequency of small cusps. Differential timing in

the termination of growth between cusps influences cusp spacing, relative cusp size, and

the presence of lophs between cusps.

Cusp shape characters are also influenced by variation in the relative growth of

the epithelium and mesenchyme that affect the pointedness or bluntness of cusps, and the

convexity of the cusp walls. Configuration characters are influenced by differences in the

spatial organization of cusps prior to initiation, in association with secondary enamel

knots, and beginning with the first forming cusps and propagating along the crown. Thus

there should be correlation between mesial and distal cusp configurations (Jernvall and

Jung, 2000). Examples of this are the positions of the hypocone and paraconid and the

25

orientation of the cristid obliqua. So if cusp pattern is the product of a dynamic

developmental program and the same developmental processes may relate aspects of

individual cusp shape and size, the independence of certain types of tooth crown

characters becomes doubtful. However, such conclusions are based on murine studies

that have some major limitations when extrapolated to other mammal groups. Mice do

not have successional sets of teeth, they have one incisor in each half of each dental

arcade that is continuously growing, and they have a diastema in place of premolars.

Researchers interested in the evolution of extant and extinct primate dentitions are

beginning to incorporate developmental data into reinterpretations of the growth

processes responsible for the variation present in primate dental patterning (Jernvall and

Jung, 2000), as well as in reassessments of the validity of dental characters historically

used in primate phylogenetics (McCollum and Sharpe, 2001). In this thesis I examine the

presence and variation of EDJ dental traits, such as accessory cusps, within the context of

this developmental paradigm.

Taxonomy of the common chimpanzee

Two species of chimpanzee are commonly recognized: Pan paniscus and Pan

troglodytes. Pan paniscus, also referred to as the bonobo or pygmy chimpanzee, is found

in the Democratic Republic of the Congo and its range is confined by the Congo River

that serves as geographic barrier (Kortland, 1995). The species distinction between Pan

paniscus and Pan troglodytes has been supported by both morphological studies (e.g.,

Johanson, 1974; Shea et al., 1993; Uchida, 1996, Pilbrow, 2006a and references therein)

and by molecular studies (Ruvolo et al., 1994; Morin et al., 1994; Won and Hey, 2005;

26

Becquet et al., 2007). The commonly recognized subspecies of Pan troglodytes include:

Pan troglodytes verus (western chimpanzees separated by the Dahomey gap), Pan

troglodytes vellorosus (Nigerian chimpanzees separated by the Sanaga River), Pan

troglodytes troglodytes (central chimpanzees separated by the Ubangi River), and Pan

troglodytes schweinfurthii (eastern chimpanzees separated by the Ubangi River and

Congo River).

While the subspecies distinction of each of these taxa is debated (Fischer et al.,

2006) and is more strongly supported for some taxa (e.g., P. t. verus) than others (e.g., the

distinction between P. t. troglodytes and P. t. schweinfurthii), evidence supporting their

distinction is both morphological (Johanson, 1974; Shea et al., 1993; Uchida, 1996,

Pilbrow, 2003, 2006a) and molecular (Morin et al., 1994; Stone et al., 2002; Won and

Hey, 2005; Gonder et al., 2006; Becquet et al., 2007). Both morphological and genetic

evidence suggest that P. t. verus is the most distinctive (either due to earlier genetic

isolation or smaller effective population size) of the other three subspecies. Genetic

diversity is relatively low for P. t. verus and P. paniscus compared to P. t. troglodytes

which is shows the highest genetic diversity (Becquet et al., 2007). Due to

methodological differences current molecular based estimates for the divergence time

between Pan species and subspecies differ among studies. Based on two recent analyses a

cautious estimate for the divergence of P. paniscus and P. troglodytes is 1.5 – 1.0 Ma,

and between P. t. verus and the central and eastern subspecies is 0.9 -0.5 Ma (Won and

Hey, 2005; Becquet et al., 2007).

Molar morphology of southern African fossil hominins

27

In this thesis I compare the molar morphology of two southern African fossil

hominins: Australopithecus africanus and Paranthropus robustus. While the generic

distinction between these taxa is not central to the topic of this thesis it is adopted

following the arguments for a both a grade and clade distinction between these taxa (see

Wood and Constantino, 2007 and references therein). Australopithecus africanus is well

represented by craniodental (and some postcranial) fossils from the sites of Sterkfontein,

Makapansgat, Taung and Gladysvale, South Africa. These fossils date to between 3.0 and

2.4 Ma. Paranthropus robustus fossils derive from the sites of Kromdraai, Swartkrans,

Gondolin, Drimolen, and Cooper’s Cave, South Africa, which are dated to 2.0 – 1.5 Ma.

The following is a review of those aspects of molar crown morphology that distinguish

the two taxa.

Robinson (1956) observed that mandibular molars are larger in P. robustus than

in Au. africanus and exhibit a size gradient of M1<M2<M3 in the former and M1<M2>M3

in the latter. Wood and Abbott (1983) noted a molar size gradient of M1<M2≈M3 for both

taxa and Suwa and colleagues (1994) found significant crown size difference in M1 (P.

robustus larger), but non-significant differences in the crown size of M2 and M3. Wood

and Abbott (1983) found no significant difference in crown shape (contra Robinson

[1956] who suggested that P. robustus molars are more oval in shape while Au. africanus

molars are more rectangular). In an analysis of cusp areas Wood and colleagues (1983)

noted that M1s of P. robustus and Au. africanus differed significantly in the relative size

of the protoconid, metaconid and hypoconulid, while M2s and M3s exhibited significant

relative size difference only in the hypoconulid. They argued that robust taxa trend

towards a reduction of the protoconid and metaconid and an increase in the entoconid and

28

hypoconulid in M1 and M2, and that fissure pattern is similar between taxa at each molar

position. Suwa and colleagues (1994) found that P. robustus tended to exhibit a relatively

smaller protoconid area (M1, M2 and M3), a relatively larger hypoconulid (M1), and

relatively larger entoconid (M2). Robinson (1963, cited in Wood et al., 1983) also

suggested that P. robustus teeth are low cusped compared to Au. africanus. Grine (2004)

documented considerable overlap in mesiodistal and buccolingual diameters of upper and

lower molars between Au. africanus and P. robustus/crassidens.

With regard to discrete dental traits Robinson (1956) noted a tendency for well-

developed protoconidal cingulum in Au. africanus that is not well-developed in P.

robustus. Wood and Abbott (1983) noted a greater incidence of protostylid expression in

P. robustus, but more marked expression when present in Au. africanus. Hlusko (2004)

found significantly different frequency distributions (although Bonferonni correction

eliminated its statistical significance) of protostylid expression between Au. africanus and

P. robustus. She noted that Au. africanus tends toward a bimodal distribution, with either

no expression or a high degree of expression, while P. robustus is the opposite with an

approximately normal distribution of protostylid expression, and consequently, that it is

frequency distribution that separates the two taxa and not a central tendency. Guatelli-

Steinberg and Irish (2005) also found that Au. africanus exhibited greater expression

protostylid (LM1). It is worth noting that Robinson (1956) considered that treatment of

the protostylid as a unit character (Dahlberg, 1950; Hlusko, 2004) was not supported due

to the presence of a cingulum farther back on the buccal surface as well as on the whole

mesial face of the crown. Greater frequency (Robinson, 1956; Wood and Abbott, 1983,

Guatelli-Steinberg and Irish, 2005) and relative size (Suwa et al., 1994) of C6 in molars

29

of P. robustus compared to those of Au. africanus has been noted as well as a higher

incidence of C7 of Au. africanus (Wood and Abbott, 1983; Guatelli-Steinberg and Irish,

2005). Irish and Guatelli-Steinberg (2003) found a high frequency of a MTC on LM1

(but check frequency) in Au. africanus. Robinson (1956) noted that Au. africanus can

exhibit a double anterior fovea while only a single fovea is present in P. robustus and a

flatter slope on buccal face than lingual in Au. africanus.

Based on both quantitative and qualitative dental traits, the taxonomic distinction

between Au. africanus and P. robustus is well supported. Although for any particular

tooth, or for an unknown specimen, the reliability of linear dimensions, relative cusp

areas, or discrete trait analysis to provide a clear taxonomic affiliation is limited. One of

the goals of this thesis is to explore whether comparisons of the detailed shape of the EDJ

would provide a more reliable means of determining the taxonomic affinities of fossil

hominin teeth.

Materials

The complete list of molar teeth used in each analysis of this thesis are given in

Appendix A. A number of teeth are used in multiple analyses, and they are identified in

the Appendix. The sample includes P. paniscus (Pp) and two subspecies of P. troglodytes

(Pt), P. t. troglodytes (Ptt) and P. t. verus (Ptv). The Ptt sample derives from the Museum

für Naturkunde (denoted as ZMB) in Berlin, Germany and the subspecies designation is

based on localities (located in Cameroon or Gabon) from which the specimens originate.

The Ptv sample derives from a skeletal collection housed at the Max Planck Institute for

Evolutionary Anthropology in Leipzig, Germany (MPI-EVA). The collection derives

30

from naturally deceased individuals collected within the research mandate of the Taï

Chimpanzee Project based in the Taï National Park, Republic of Côte d'Ivoire, and only

Ptv is present in this area. The Pp sample derives from the Royal Museum for Central

Africa in Tervuren, Belgium (MRAC). Species designation is based on locality

information and museum catalogue information associated with each specimen. Nine

additional individuals from the Museum für Naturkunde and two from the Royal Museum

for Central Africa are either captive specimens, or do not have locality information. All

molars are from associated mandibles from which the tooth type is determined (e.g., first

or second mandibular molar).

The Gorilla g. beringei specimens derive from mandibles curated at the National

Museum of Natural History (NMNH), Smithsonian Institution, Washington, USA. They

were collected in Rwanda and their taxonomic affiliation is based on museum records.

The three Pongo pygmaeus molars are curated at ZMB and their taxonomic affiliation,

when available, is based on museum records. Two modern human molars also come from

the NMNH, but they are from a collection of isolated specimens and therefore their tooth

type is not certain and they are considered either first or second molars. Two modern

human molars derive from mandibles belonging to Bronze Age burials. One is curated by

Headland Archaeology Ltd (HA), Edinburgh, Scotland and the other by the Hull and East

Riding Museum (HE), Hull, UK.

The fossil hominin sample includes Au. africanus molars from the site of

Sterkfontein Member 4 (Moggi-Cecchi et al., 2006) and P. robustus molars from the sites

of Swartktrans, Gondolin and Drimolen (Robinson, 1956; Tobias et al., 1977; Grine

1989; Menter et al., 1999; Keyser et al., 2000; De Ruiter, pers. comm. 2006). These

31

specimens are curated at the Transvaal Museum (TM), Northern Flagship Institution,

Pretoria and The University of Witwatersrand (UW), Johannesburg, South Africa. The

taxonomic affiliation and tooth type for each specimen were determined from the above

references. Four molars belonging to H. neanderthalensis derive from the site of El

Sidron, Spain (Rosas et al., 2006) and are curated at the Museo Nacional de Ciencias

Naturales (MNCN), Madrid, Spain. The single Gigantopithecus blacki specimen is

curated at the Senckenberg Forschungsinstitut und Naturmuseum (SFN), Frankfurt,

Germany and was collected by von Koenigswald from a Chinese drugstore in Hong Kong

(Weidenreich, 1945; von Koenigswald, 1952). This tooth is listed as a third molar

(Specimen 4) by von Koenigswald (1952), however, it is not clear what the criteria were

for this designation. Given the divergent distal root I believe it is possible that this molar

is a second molar from an immature individual.

Methods

Many of the methods employed were used in multiple analyses and are discussed

in each chapter as appropriate. In this section I discuss a number of methodological issues

in greater detail than in specific chapters as the chapters were written in manuscript form,

which necessitates a limited discussion of methodological issues in some cases. The

methods discussed below include 1) micro-computed tomography, 2) tissue segmentation

and surface generation protocols, and 3) geometric morphometric analyses, and 4)

visualization of EDJ shape differences.

Micro-computed tomography

32

Microcomputed tomography of dental specimens was completed using two

tomographic scanners. The first system is a SKYSCAN 1172 Desktop Scanner and the

following scan parameters were used: 100 Kv, 94 mA, 2.0mm aluminum and copper

filter, 0.12 – 0.15 rotation step, 1024 x 1024 or 2048 x 2048 matrix, 360 degrees of

rotation, 2 frame averaging. Raw projections were converted into TIFF image stacks

using NRecon (parameters: ring artifact correction = 10; beam hardening = 30%). Pixel

dimensions and slice thickness between reconstructed serial images were isometric with

resolutions ranging between 14 and 28 microns (µm) (e.g., isometric voxels of 14 µm ×

14 µm × 14 µm to 28 µm × 28 µm × 28 µm).

The second system is a SCANCO µCT40 and the following scan parameters were

used: 70 Kv, 114 µA, 0.36 rotation step, 1024 x 1024 matrix, 180 degrees of rotation, no

frame averaging. Pixel dimensions and slice spacing of the resultant images were 16µm.

In some cases image stacks were downsampled from their original resolution in order to

reduce the size of the data and facilitate processing of the tomographic scans. The effect

of downsampling on the resulting analyses of EDJ shape is addressed below.

Tissue segmentation and surface generation protocols

To facilitate tissue segmentation, the complete image stack for each tooth was

filtered using a three-dimensional median filter (kernel size of 1 or 3) followed by a mean

of least variance filter (kernel size of 1 or 3), implemented as a computer-programmed

macro. This filtering process results in more homogenous tissue classes (e.g., enamel vs.

dentine) and allocates pixels with intermediate gray-scale values at tissue interfaces (i.e.,

air-enamel, enamel-dentine, air-dentine) to the appropriate tissue (Schulze and Pearce,

33

1994). The effect of filtering on the resulting analyses of EDJ shape is addressed below.

The choice of kernel size was dictated by the degree of contrast between enamel and

dentine tissue. When contrast is high (i.e., the range of gray-scale values representing

each tissue are distinct and with little overlap) a kernel size of one is used. When contrast

is low a kernel size of three is used in order to reduce the need for significant amounts of

manual segmentation.

Filtered image stacks were imported into the Amira software package (v4.1,

www.amiravis.com) and enamel and dentine tissues were segmented using the 3D voxel

value histogram and its distribution of gray-scale values, which typically presents a

trimodal distribution with one peak representing dentine, another peak representing

enamel, and a third peak representing air and background noise in the image stack. In

fossil teeth where the enamel and dentine differ substantially in their degree of

mineralization (and therefore their densities, and thus also in the ability of X-rays to pass

through them), the filtering process results in gray-scale pixel value distributions for each

tissue that do not overlap. In other teeth, diagenetic alteration (e.g., dentine

remineralization) may result in similar tissue densities and thus overlapping gray-scale

pixel value ranges for enamel and dentine (Olejniczak and Grine, 2006). Even after

filtering, there is often an incomplete separation between the two, and a decision must be

made about the range of gray-scale values allocated to each tissue. Only teeth in which

there was a clear separation of enamel and dentine, resulting in well-distinguished gray-

scale values and accurate representations of the EDJ, were included. Operationally, this

meant that if the tooth could not be segmented using a filter kernel size of 1 or 3 it was

34

removed from the study as larger kernel sizes prevent accurate representations of the EDJ

and OES.

After segmentation, the enamel cap and EDJ are reconstructed as triangle-based

surface models using Amira (surface generation module using unconstrained smoothing

parameter). The OES and EDJ of a chimpanzee molar are illustrated in Fig. 1.1. Small

portions of the enamel cap or EDJ are missing in some teeth, or are damaged due to

cracks, and in these cases the defects were corrected digitally using the software

Geomagic Studio 10 (www.geomagic.com). Teeth that show evidence of significant

damage or missing areas were excluded from the study. In a few teeth that were

minimally worn the tips of the dentine horns were repaired using the “Fill Holes” feature

in Geomagic Studio 10. In specimens that preserved only the enamel cap, a surface model

of the EDJ was created by digitally removing the occlusal surface of the reconstructed

enamel cap surface model.

Geometric morphometric analysis protocols

The GM analysis of EDJ shape involves a number of steps that are outlined in

detail below. These steps can be summarized as 1) the collection of anatomical

landmarks, 2) the derivation of a homologous set of landmarks for each specimen; 3)

Procrustes superimposition of the landmarks of each specimen, 4) statistical analyses of

shape variation within and between groups, and 5) visualization of shape variation within

and between groups.

Collection of anatomical landmarks. EDJ surface models (PLY format) were

imported into Amira for the collection of the Cartesian coordinates of three sets of

35

anatomical landmarks (see Chapter 2, Fig. 2.1). The first set (referred to as “MAIN”)

included eight landmarks: one on the tip of the dentine horn of each primary cusp [i.e.,

protoconid (1), metaconid (2), entoconid (3), hypoconid (4) and hypoconulid (5)], one at

the lowest point on the crests connecting, respectively, the protoconid and metaconid (6),

the protoconid and hypoconid (7), and the hypoconid and hypoconulid (8). The second

set (referred to as the “RIDGE” curve) includes landmarks (approximately 50-70) along

the top of the ridges running between each of the five dentine horns. This set of

landmarks forms a closed ellipse, beginning at the tip of the protoconid and proceeding

anticlockwise in a lingual direction. In the case of teeth with accessory cusps (e.g., cusp 6

or cusp 7) these dentine horns were ignored and landmarks were collected on either side

of the feature. The third set (referred to as the “CERVIX” curve) includes landmarks

(approximately 40-50) along the cervix, or cemento-enamel junction, of the tooth crown.

This set of landmarks also forms a closed ellipse, beginning on the cervix below the

protoconid dentine horn and proceeding anticlockwise in a lingual direction. Where small

fragments of enamel were missing at the cervix its location was estimated. Landmark

datasets were exported as text files.

Derivation of homologous landmarks. For each specimen a single set of

homologous landmarks and semilandmarks is derived from the three landmark files

discussed above. For both the RIDGE and CERVIX curve landmark sets a smooth curve

is interpolated using a cubic spline function (note: a cubic spline is used so that the curve

is forced to pass through each measured coordinate). Interpolated curves are then

resampled to achieve identical point counts between specimens – these resampled points

were used as semilandmarks. In the case of the RIDGE curve the eight MAIN landmarks

36

are projected onto the curve dividing it into eight sections. For each section a large

sample of very closely spaced points are computed along the curve and the distances

between adjacent points are calculated and summed to approximate the length along the

curve between the MAIN landmarks. Each length is divided by a given number and the

coordinate location at each equally spaced distance is recorded. The number of divisions

between the MAIN landmarks is: ten between #2 and #3 and #3 and #5, seven between

#2 and #6 and #1 and #6, five between #1 and #7 and #7 and #4, four between #4 and #8

and #8 and #5. In the case of the cervix curve, its length is calculated in the same way

and 70 equidistantly spaced points are derived.

These equidistantly spaced points are then used as starting positions for the

semilandmark algorithm: semilandmarks were iteratively allowed to slide along their

respective curves (i.e., RIDGE curve [N =60 including the eight MAIN landmarks] and

CERVIX curve [N = 70]) to minimize the bending energy of the thin-plate spline

interpolation function computed between each specimen and the sample Procrustes

average. We used the algorithm of Bookstein (1997; Gunz et al., 2005) that allows points

to slide along tangents to the curve. These tangents were approximated for each

semilandmark by the vector between the two neighboring points. Only the tips of the

dentine horns were used as fixed landmarks, all other points were treated as

semilandmarks. After sliding these semilandmarks are considered homologous for the

purpose of multivariate analyses.

Procrustes superimposition. The homologous set of landmarks and semilandmarks

is converted to shape coordinates by Generalized Least Squares Procrustes

superimposition (Gower, 1975; Rohlf and Slice, 1990). This removes information about

37

location and orientation from the raw coordinates and standardizes each specimen to unit

centroid size; a size-measure computed as the square root of the sum of squared

Euclidean distances from each landmark to the specimen’s centroid (Dryden and Mardia,

1998). All data preprocessing was done in Mathematica v6.0 (www.wolfram.com) using

a software routine written by Philipp Gunz.

Analysis of EDJ shape. Two statistical methods were used to assess EDJ shape

variation between taxon and tooth type in chapters two and three. A principal components

analysis (PCA) of shape coordinates (Bookstein, 1991; Rohlf, 1993) after Procrustes

superimposition is an eigen-decomposition of the variance-covariance matrix of

Procrustes coordinates. It creates a set of hypothetical variables (principal components or

PC), that are linear combinations of the original variables. Beginning with the first PC, it

produces orthogonal axes which represent major aspects of shape variation (Hammer and

Harper, 2006).

A canonical variates analysis (CVA) was used to assess the degree of EDJ shape

difference between taxa and tooth type and to identify those aspects of shape that best

separate groups. In each case the CVA was computed as linear discriminants in the

software package R (www.r-project.org) in the subspace of the first few principal

components (i.e., using only a subset of all PCs) of the Procrustes shape coordinates.

Projecting into a lower dimensional subspace was necessary because the number of

variables (i.e., landmarks) greatly exceeded the sample size (i.e., molar teeth); using too

many variables to compute the discriminant axes can result in an unrealistic and unstable

degree of discrimination. The decision as to the number of PCs to incorporate into the

CVA was guided by a protocol in which the specimen labels were randomized and the

38

resulting CVA was assessed with different numbers of PCs included. The number of PCs

included (usually 5-10) did not exceed the point at which randomized labels began to

exhibit clustering patterns in the resulting CVA. In other words, it was confirmed that

there was no separation between randomly relabeled groups. This phenomenon of

clustering in a randomized dataset is illustrated in Fig. 1.2. A CVA of original second

molar data using 6 PCs exhibits marked separation among the groups. With randomized

data, clustering appears with the inclusion of an increasing number of PCs.

The accuracy with which EDJ shape correctly classifies molars, according to

species and tooth type, was tested by cross validating the canonical variates analysis. In

this process each specimen in turn was considered unclassified and then classified by all

the others using posterior probabilities. Classification was implemented in R with groups

assigned equal prior probabilities.

Visualization of EDJ shape change

An advantage of the geometric morphometric methodology employed to compare

EDJ morphology is that differences in EDJ shape can be visualized in 3D using the

quantitative results of the statistical analyses. To do so, an EDJ surface model can be

deformed with respect to changes in shape among specimens in the study sample (Gunz

and Harvati, 2007). This method is employed in two ways in this thesis. First, it is used to

visualize the mean shape of groups (e.g., the mean shape of the first molar of Au.

africanus compared to the mean shape of the first molar of P. robustus). Second, it is

used to visualize shape differences along canonical variate axes that separate species

and/or molar type (chapter three). The first step involves the creation of a generic EDJ

39

surface model. This was accomplished by collecting several thousand landmarks in

Amira on the EDJ of one molar of the study sample (either a Pan molar or a hominin

molar). These points were converted to a triangulated surface model (PLY file format)

using Geomagic Studio 10 (wrap module). The thin-plate spline interpolation function

between the landmarks of this specimen and the appropriate mean configuration

Procrustes shape space was used to bring this surface model into Procrustes shape space.

Finally, a thin-plate spline interpolation was computed to morph this generic surface

model (now in Procrustes shape space) into a target form. For the visualization of the

mean shape of a particular group the target form was the group average of Procrustes

shape coordinates. For the visualization of a CVA axis the generic surface model (now in

Procrustes shape space) was morphed by regressing all Procrustes shape coordinates on

the respective CVA scores and adding a scaling factor.

The effect of downsampling image resolution on EDJ surface morphology

In most cases in the study sample, teeth were downsampled from an original

resolution of ~14 µm voxel resolution to ~30 µm voxel resolution in order to reduce the

amount of data to a more manageable level and to speed up the manual segmentation of

some specimens. I tested the effect of downsampling on both qualitative and quantitative

analyses in order to ensure that this source of variation was unlikely to influence the

results of my analyses. To do so one chimpanzee tooth was downsampled to three

different resolutions in Amira using the resample function (Triangle filter). This resulted

in four image stacks with resolutions of 14 µm, 28 µm, 50 µm and 100 µm. Each image

stack was filtered using a 3D median filter (kernel size = 1) and a mean of least variance

40

filter (kernel size = 1) to facilitate tissue segmentation. From each segmented stack a

surface model of the EDJ was reconstructed (Fig. 1.3).

To test the influence of downsampling on the geometric morphometric analysis of

EDJ shape a landmark data set (as discussed above) was collected on each EDJ surface.

These four data sets were included in an analysis of EDJ shape as individual specimens,

along with other chimpanzee teeth. Figure 1.4 presents the resulting PCA plot of the first

and second principal components. As can be seen from this figure downsampling can

influence the results of such analysis, particularly when the degree of downsampling

exceeds a factor of two (e.g., represented by the 50 µm and 100 µm models). This is

likely due to the differences in the height of the dentine horns and the location of the

cervix which are altered with increasingly downsampled data.

For the qualitative test the EDJ surfaces generated from each resolution were

compared with respect to pertinent aspects of shape (e.g., dentine horn shape and size of

the main cusps, crest morphology, accessory cusp dentine horn morphology, and cervix

position). There are only minor alterations in the morphology of the EDJ between the 14

µm and 28 µm surface models. The dentine horns of the primary cusps are similar in their

degree of pointedness, those of the accessory cusps (in this case a double C6) are still

identifiable, and the crests on the slopes of the dentine horns are similar in their degree of

expression. The 50 µm surface model exhibits blunter dentine horns on both primary and

accessory cusps and crests which are less pronounced. The 100 µm surface model is

markedly different in its qualitatively assessed shape from the original EDJ surface.

Dentine horn morphology is substantially altered, the crest morphology is absent within

the occlusal basin and the mesial dentine horn of the double C6 is absent. The results of

41

this assessment suggest that accurate evaluation of qualitative morphology of the EDJ of

hominoid teeth requires a voxel resolution no less ~50 µm and preferably less than 30

µm.

The effect of filtering microCT images on EDJ surface morphology

Filtering of microCT images is a widely accepted method to increase the signal to

noise ratio within the image stacks and to facilitate tissue segmentation. Filtering does,

however, alter the original data and therefore it was necessary to assess the affect of

filtering on the data collected in this thesis. To do so a single chimpanzee tooth (voxel

size of 14 µm) was assessed with no filtering, with a 3D median filter and mean of least

variance filter each having a kernel size of one, and with a 3D median filter and mean of

least variance filter each having a kernel size of three. For each filtered data set the

dentine tissue was segmented and the EDJ surface reconstructed.

To test the influence of filtering on the geometric morphometric analysis of EDJ

shape a landmark data set was collected on each EDJ surface. These three data sets were

included in an analysis of EDJ shape as individual specimens, along with other

chimpanzee teeth. Figure 1.4 presents the resulting PCA plot of the first and second

principal components. As can be seen from this figure filtering with kernel sizes of one or

three has little influence the results position of the specimen in Procrustes shape space

and thus little influence on EDJ shape as captured by landmarks.

To test the influence of filtering on qualitatively assessed aspects of EDJ shape

(e.g., dentine horn shape and size of the main cusps, crest morphology, accessory cusp

dentine horn morphology, and cervix position) surface models resulting from each filter

42

combination were compared with a model derived with no filtering to the original data.

As can be seen from Fig. 1.5 variation in the filter settings from no filtering, to kernel

sizes of one and three has little affect on the morphology that is of interest for this thesis.

The morphology of dentine horns and crest features is not altered significantly even when

a filter size of 3 is used. Thus scoring of discrete traits involving accessory cusps and

crests will not be significantly altered by the use of image filtering.

43

Figure 1.1. Surface reconstructions of the A) outer enamel surface and B) enamel-dentine junction. The major cusps on the OES and dentine horns on the EDJ are labeled.

44

Figure 1.2. These CVA plots illustrate the potential problem of including a large number of PCs in a CVA analysis. A) CVA of original second molar data using 6 PCs showing strong separation between groups. With randomized data, which should exhibit no clustering, clustering is achieved with the inclusion of an increasing number of PCs. B) – F) CVAs based on 4, 6, 8, 10, 20 PCs respectively. The degree of overlap in (C) supports the use of 6 PCs in the original analysis.

45

Figure 1.3. Illustration of the affect of downsampling the resolution of microCT image stacks on the resulting surface reconstruction of the EDJ. A) original 14 µm voxel size; B) 28 µm; C) 50 µm; D) 100 µm.

46

Figure 1.4. Principal component analysis assessing the influence of downsampling and filtering on the analysis of EDJ shape. Downsampling does not have a significant influence on Procrustes distances until resolutions reach 50 microns and greater. There is little influence on Procrustes distance either not using a filter or when the filter kernel size is 1_1 or 3_3.

47

Figure 1.5. Illustration of the affect of filtering on the resulting surface reconstruction of the EDJ. A) No filtering; B) median and mean of least variance (MLV) with a kernel size of 1; C) median and mean of least variance with a kernel size of 3.

48

CHAPTER 2: DISCRIMINATION OF SPECIES AND SUBSPECIES OF PAN

USING THE EDJ MORPHOLOGY OF LOWER MOLARS

Abstract

Previous research has demonstrated that species and subspecies of Pan can be

distinguished on the basis of the shape of their molar crowns. Thus, there is potential for

fossil hominin taxa to be distinguished at similar taxonomic levels, but because of

occlusal attrition the surface morphology of tooth crowns is often absent in fossil teeth.

The enamel-dentine junction (EDJ) of molar teeth possesses considerable information

regarding the original shape of the tooth and is preserved longer than the outer enamel

surface (OES). In this study we investigate whether the shape of the EDJ of lower first

and second molars can distinguish species and subspecies of Pan. Micro-computed

tomography is employed to non-destructively image the EDJ and geometric

morphometric analytical methods are used to compare EDJ shape among samples of Pan

paniscus (N = 17), Pan t. troglodytes (N = 14), and Pan t. verus (N = 18). The

classification of specimens of unknown taxonomic affiliation (N = 13) is also assessed.

Results indicate that EDJ morphology successfully distinguishes among both species and

subspecies of Pan and can support the designation of unknown specimens to both taxon

and molar type. Morphological differences in EDJ shape among taxa are subtle and relate

to the relative height and spacing of the dentine horns, the height of the dentine crown,

and the shape of the crown base.

49

Introduction

Molar crown morphology has been used to address taxonomic questions in extant

apes (Johanson, 1974; Hartman, 1988; Uchida, 1992, 1996, 1998a,b; Pilbrow, 2003,

2006a,b) and fossil hominins (e.g., Robinson 1956; Sperber, 1974; Wood and Abbott,

1983; Wood et al., 1983, Wood, 1991; Suwa et al., 1994). The taxonomic distinctiveness

of molar crown shape has most recently been demonstrated by Pilbrow (2003, 2006a)

who, based on a large number of linear crown measurements, was able to distinguish

between species, subspecies and even populations of African apes (particularly

chimpanzees).

The description and analysis of newly discovered hominin fossil teeth has

traditionally been limited to gross linear dimensions (e.g., buccolingual and mesiodistal

diameter of the crown). This is understandable because in a partially worn fossil tooth not

many aspects of shape can be measured and defended as being homologous. More

recently measurements such as cusp surface areas and crown base shape have improved

the discrimination of taxa, but considerable overlap often remains, limiting the taxonomic

level at which closely related taxa can be distinguished. Unfortunately, the availability of

these linear and areal measurements on worn fossil teeth is limited by attrition, and this

limitation restricts our ability to apply analytical methods which incorporate the

complexity of the shape of the OES.

It has long been acknowledged that the enamel-dentine junction (EDJ), which

underlies the enamel cap of primate teeth, carries information about the original shape of

the tooth crown (Kraus, 1952; Korenhof, 1960, 1961, 1982; Nager, 1960; Kraus and

Jordan, 1965; Sakai et al., 1965, 1967a,b, 1969; Sakai and Hanamura, 1971, 1973a,b;

50

Corruccini, 1987a,b, 1998; Schwartz et al., 1998; Sasaki and Kanazawa, 1999; Skinner et

al., 2008) and that it can be used as a source of taxonomically-relevant data (Corruccini,

1998; Olejniczak et al., 2004, 2007; Macchiarelli et al., 2006; Suwa et al., 2007; Skinner

et al, nd). Furthermore, it is homologous among teeth and taxa, provides considerable

scope for capturing detailed information about tooth shape, and is preserved throughout

the initial stages of tooth wear (and longer in thicker-enameled, relatively bunodont

lower-cusped hominin taxa).

The goal of this project is to assess the taxonomic distinctiveness of the EDJ

morphology of lower molars in species and subspecies of Pan. If it can be demonstrated

that the Pan EDJ can distinguish these taxonomic levels, then it is likely that the same

method will have utility for discriminating the species and subspecies of the other genera

within the Pan/Homo clade.

Chimpanzee taxonomy

Two species of chimpanzee are commonly recognized: Pan paniscus and Pan

troglodytes. Pan paniscus, also referred to as the bonobo or pygmy chimpanzee, is found

in the Democratic Republic of the Congo and its range is confined by the Congo River.

The species distinction between P. paniscus and P. troglodytes has been supported by

both morphological (e.g., Coolidge, 1933; Johanson, 1974; Shea et al., 1993; Uchida,

1996, Pilbrow, 2006a and references therein) and molecular studies (Ruvolo et al., 1994;

Morin et al., 1994; Won and Hey, 2005; Becquet et al., 2007).

There are a number of commonly recognized subspecies of Pan troglodytes

whose ranges are separated by geographic barriers: Pan troglodytes verus (western

51

chimpanzees separated by the Dahomey gap), Pan troglodytes vellorosus (Nigerian

chimpanzees separated by the Sanaga River), Pan troglodytes troglodytes (central

chimpanzees separated by the Ubangi River), and Pan troglodytes schweinfurthii (eastern

chimpanzees separated by the Ubangi River and Congo River). While the subspecies

distinction of each of these taxa is debated (Fischer et al., 2006) and is more strongly

supported for some taxa (e.g., P. t. verus) than others (e.g., the distinction between P. t.

troglodytes and P. t. schweinfurthii) evidence supporting their distinction is both

morphological (Johanson, 1974; Shea et al., 1993; Uchida, 1996, Pilbrow, 2003, 2006a)

and molecular (Morin et al., 1994; Stone et al., 2002; Won and Hey, 2005; Gonder et al.,

2006; Becquet et al., 2007). Both morphological and genetic evidence suggest that P. t.

verus is the most distinctive (either due to earlier genetic isolation or smaller effective

population size) from the other three subspecies.

Hypotheses

Based on the results of previous studies (reviewed above) that have demonstrated

the distinctiveness of molar crown shape among species and subspecies of Pan, and the

evidence that the EDJ contributes significantly to crown shape (Korenhof, 1960; Kraus

and Jordan, 1965; Corruccini, 1987a, 1998) we test two hypotheses. First, whether EDJ

morphology of molar tooth crowns successfully distinguishes between species and

subspecies of chimpanzee. Second, whether the subspecies of P. troglodytes are more

similar in shape to each other than either is to P. paniscus and support phylogenetic

relationships based on morphological and genetic data. Support for these hypotheses

would suggest that the factors which have lead to divergence in tooth shape between taxa

52

act on the processes of early tooth development when the shape of the EDJ is being

established. Lack of support for these hypotheses would suggest that EDJ morphology

carries a more conservative taxonomic and phylogenetic signal and that differences in

enamel growth are responsible for the findings of previous studies of the shape of the

enamel surface.

In order to test these hypotheses we performed a geometric morphometrics

analysis of anatomical landmarks collected on the surface of the EDJ. The EDJ surface of

each molar was non-destructively imaged using micro-computed tomography.

Anatomical landmarks on the EDJ were chosen to capture the overall crown shape of the

dentine crown, including crown height, dentine horn height, dentine horn spacing and the

shape of the cervix. This methodology not only allows the quantitative assessment of

shape differences among taxa, but it also allows useful visual depictions of the shape

differences that distinguish taxa. The first hypothesis was tested by assessing the

accuracy with which molars were correctly classified to their known taxonomic

affiliation and the second was tested by comparing the phenetic similarity between taxa

against phylogenetic expectations based on molecular data. As for the teeth whose

taxonomy is not so well established, for some of them there is strong circumstantial

evidence (where and by whom they were recovered) and this information can be

compared with the taxonomic affinities suggested by the EDJ morphology.

Materials and Methods

Study sample

53

The first and second mandibular molars used in the study sample are listed in

Tables 2.1 and 2.2, respectively. The sample includes P. paniscus (Pp) and two

subspecies of P. troglodytes (Pt), P. t. troglodytes (Ptt) and P. t. verus (Ptv). The Ptt

sample derives from the Museum für Naturkunde (denoted as ZMB) in Berlin, Germany

and the subspecies designation is based on localities (located in Cameroon or Gabon)

from which the specimens originate. The Ptv sample derives from a skeletal collection

housed at the Max Planck Institute for Evolutionary Anthropology in Leipzig, Germany

(MPI). The collection derives from naturally deceased individuals collected within the

research mandate of the Taï Chimpanzee Project based in the Taï National Park, Republic

of Côte d'Ivoire. The subspecies designation is based on the fact that only Ptv is present

in this area. The Pp sample derives from the Royal Museum for Central Africa in

Tervuren, Belgium (MRAC). Species designation is based on locality information and

museum catalogue information associated with each specimen.

Nine additional individuals from the Museum für Naturkunde and two from the

Royal Museum for Central Africa are either captive specimens or do not have locality

information (Table 2.3). These specimens are treated as having an unknown taxonomic

affiliation in order to compare their morphology to specimens of the other taxa. All

molars are from associated mandibles from which tooth type is determined (e.g., first or

second mandibular molar).

Microcomputed tomography

Each tooth was microCT scanned using a SKYSCAN 1172 Desktop Scanner

(100Kv, 94mA, 2.0mm aluminum and copper filter, 0.12 rotation step, 360 degrees of

54

rotation, 2 frame averaging). Raw projections were converted into TIFF image stacks

using NRecon (parameters: ring artifact correction = 10; beam hardening = 30%). Pixel

dimensions and slice spacing of the resultant images ranged between 10 – 20

micrometers, or microns (µm). To reduce the size of the resulting files, teeth were

downsampled to a resolution of 30 µm using Amira (Triangle filter).

EDJ surface reconstruction

To facilitate tissue segmentation, the complete image stack for each tooth was

filtered using a three-dimensional median filter (kernel size of 3) followed by a mean of

least variance filter (kernel size of 3), implemented as a computer-programmed macro.

This filtering process results in more homogenous tissue classes (e.g., enamel vs. dentine)

and allocates pixels with intermediate gray-scale values at tissue interfaces (i.e., air-

enamel, enamel-dentine, air-dentine) to the appropriate tissue (Schulze and Pearce, 1994).

Filtered image stacks were imported into the Amira software package (v4.1,

www.amiravis.com) and enamel and dentine tissues were segmented using the 3D voxel

value histogram and its distribution of gray-scale values, which typically presents a

trimodal distribution with one peak representing dentine, another peak representing

enamel, and a third peak representing air and background noise in the images. Only teeth

in which there was a clear separation of enamel and dentine, resulting in well-

distinguished gray-scale values and accurate representations of the EDJ, were used in the

study.

After segmentation, the EDJ is reconstructed as triangle-based surface model

using Amira (surface generation module using unconstrained smoothing parameter).

55

Small portions of the EDJ are missing in some teeth, and in these cases the defects were

corrected digitally using the software Geomagic Studio v.10 (www.geomagic.com).

Teeth which showed evidence of significant damage or missing areas were excluded from

the study. In a few minimally worn teeth the tips of the dentine horns were repaired. In

specimens that preserved only the enamel cap, a surface model of the EDJ was created by

digitally removing the occlusal surface of the reconstructed enamel cap surface model.

Collection of landmarks

EDJ surface models (PLY format) were imported into Amira for the collection of

three sets of 3D anatomical landmarks (Fig. 2.1). The first set (referred to as “MAIN”)

included eight landmarks: one on the tip of the dentine horn of each primary cusp [i.e.,

protoconid (1), metaconid (2), entoconid (3), hypoconid (4) and hypoconulid (5)], one at

the lowest point on the crests connecting, respectively, the protoconid and metaconid (6),

the protoconid and hypoconid (7), and the hypoconid and hypoconulid (8). The second

set (referred to as the RIDGE curve) includes 3D coordinates (approximately 50-70)

along the top of the ridges running between each of the five dentine horns. This set of

landmarks forms a closed ellipse, beginning at the tip of the protoconid and moving

anticlockwise in a lingual direction. In the case of teeth with accessory cusps (e.g., cusp 6

or cusp 7) these dentine horns were ignored and points were collected on either side of

the dentine horn. The third set (referred to as the CERVIX curve) includes 3D

coordinates (approximately 40-50) along the cervix, or cemento-enamel junction, of the

tooth crown. This set of landmarks also forms a closed ellipse, beginning below the

protoconid dentine horn and moving anticlockwise in a lingual direction. Where small

56

fragments of enamel were missing at the cervix its location was estimated. Landmark

datasets were exported as text files. We tested the influence of downsampling (from 15-

30 microns) and filtering (no filtering, kernel sizes of 1 and kernel sizes of 3) on

landmark configurations of a single specimen. The distance in Procrustes shape space

between the original specimens and those that had been downsampled or filtered with

different settings was minimal and considerably less than distances from the original

specimen to any other individuals of the same species or tooth type.

Derivation of homologous landmarks

For each specimen a single set of homologous landmarks and semilandmarks is

derived from the three landmark files discussed above. For both the RIDGE and CERVIX

curve landmark sets a smooth curve is interpolated using a cubic spline function (note: a

cubic spline is used so that the curve is forced to pass through each measured coordinate).

Interpolated curves are then resampled to achieve identical point counts between

specimens – these resampled points were used as semilandmarks. In the case of the

RIDGE curve the eight MAIN landmarks are projected onto the curve dividing the curve

into eight sections. For each section a large sample of very closely spaced points are

computed along the curve and the distances between adjacent points are calculated and

summed together to approximate the length along the curve between the MAIN

landmarks. Each length is divided by a given number and the coordinate location at each

equally spaced distance is recorded. The number of divisions between the MAIN

landmarks is: ten between #2 and #3 and #3 and #5, seven between #2 and #6 and #1 and

#6, five between #1 and #7 and #7 and #4, four between #4 and #8 and #8 and #5. In the

57

case of the cervix curve, its length is calculated in the same way and 70 equally spaced

points are derived.

These equidistantly spaced points are then used as starting positions for the

semilandmark algorithm: semilandmarks were iteratively allowed to slide along their

respective curves (i.e., RIDGE curve [N =60 including the eight MAIN landmarks] and

CERVIX curve [N = 70]) to minimize the bending energy of the thin-plate spline

interpolation function computed between each specimen and the sample Procrustes

average. We used the algorithm of Bookstein (1997; Gunz et al., 2005) that allows points

to slide along tangents to the curve. These tangents were approximated for each

semilandmark by the vector between the two neighboring points. Only the tips of the

dentine horns were used as fixed landmarks, all other points were treated as

semilandmarks. After sliding these semilandmarks are considered homologous for the

purpose of multivariate analyses.

Procrustes superimposition

The homologous set of landmarks and semilandmarks is converted to shape

coordinates by Generalized Least Squares Procrustes superimposition (Gower, 1975;

Rohlf and Slice, 1990). This removes information about location and orientation from the

raw coordinates and standardizes each specimen to unit centroid size; a size-measure

computed as the square root of the sum of squared Euclidean distances from each

landmark to the specimen’s centroid (Dryden and Mardia, 1998). All data preprocessing

was done in Mathematica v6.0 (www.wolfram.com) suing a software routine written by

PG.

58

Analysis of EDJ shape

Two statistical methods were used to assess EDJ shape variation in the study

sample: principal components analysis (PCA) and canonical variates analysis (CVA). A

PCA of shape coordinates (Bookstein, 1991; Rohlf, 1993) after Procrustes

superimposition is an eigen-decomposition of the variance-covariance matrix of

Procrustes coordinates. A CVA was computed as linear discriminants in the software

package R (www.r-project.org) in the subspace of the first few principal components

(i.e., using only a subset of all PCs) of the Procrustes shape coordinates. Projecting into a

lower dimensional subspace was necessary because the number of variables greatly

exceeded our sample size; using too many variables to compute the discriminant axes

results in an unrealistic and unstable degree of discrimination. Our decision as to the

number of PCs to incorporate into the CVA was guided by a protocol in which the

specimen labels were randomized and the resulting CVA was assessed with variable

numbers of PCs included. The number of PCs included (in these analyses 5-10) did not

exceed the point at which randomized labels began to exhibit clustering patterns in the

resulting CVA. In other words, we confirmed that we would find no separation with

randomly rearranged group labels.

The accuracy with which EDJ shape correctly classifies molars, according to

species and tooth type, was tested by cross validating the canonical variates analysis. In

this process each specimen in turn was considered unclassified and then classified by all

the others using posterior probabilities. Classification was implemented in R; groups

were assigned equal prior probabilities.

59

Visualization of EDJ shape variation

To visualize the shape variation of the EDJ between taxa and between molar types

we employed a method which allows a template EDJ surface reconstruction to be

deformed to match the mean shape of a particular group (Gunz and Harvati, 2007).

Several thousand points were collected on the EDJ surface of one specimen and

converted to a triangulated surface model using Geomagic Studio. We then used the thin-

plate spline interpolation function between the landmark configuration of this specimen

and the appropriate mean configuration in Procrustes space, to warp the vertices of this

surface into Procrustes space. Processed in this way the mean shape of each group was

visualized and compared.

Results

First molars

The PCA of Procrustes shape coordinates of the first molar sample is illustrated in

Fig. 2.2a (PC1 = 31.8% and PC2 = 16% of total variation). A CVA, based on the first

five PCs (explaining 72.3% of the total variation) of first molar dataset is presented in

Fig. 2.2b. Collectively, CV1 (65%) and CV2 (13%) separate the three taxa. The

combination of overlap in the PCA and marked separation along the first two CVs

suggests that there are consistent, but small scale, differences in shape between the taxa.

Using a cross-validation analysis of the CVA the accuracy of classification to species and

subspecies is presented in Table 2.1. Three molars are misclassified at the species level

and one molar is misclassified at the subspecies level. These results support our first

60

hypothesis regarding the taxonomic distinctiveness of EDJ shape for first molars. To test

the second hypothesis we calculate the Procrustes distance (based on the first five PCs)

between the mean first molar shape of each taxon (Table 2.4). Based on the first molars

the null hypothesis is rejected as the distance in Procrustes shape space is greater between

Ptt and Ptv than the distance between either subspecies and Pp.

The mean shape of the first molar of each taxon is illustrated in Fig. 2.3. The

mean shape of Pp M1s compared to the mean shape of the combined Pt M1 sample is

visualized in Fig. 2.4a. This represents the shape differences at the species level and

includes a relatively narrower trigonid and wider talonid (particularly in the more distal

placement of the entoconid and hypoconulid) in Pt compared to Pp and a reduction in the

relative height of the talonid dentine horns in Pt compared to Pp. The mean shape

differences in the two subspecies, Ptt and Ptv, are illustrated in Fig. 2.4b. Ptt M1s exhibit

a relatively taller dentine crown, taller dentine horns, and a narrower talonid whereas Ptv

M1s exhibit a shorter dentine crown, shorter dentine horns, and a wider talonid.

Second molars

The principal component analysis of the second molar sample is illustrated in Fig.

2.5a (PC1 = 26.9% and PC2 = 20.6%). A CVA, based on the first six PCs (explaining

75.1% of the total variation) of the second molar dataset is presented in Fig. 2.5b. As with

the analysis of first molars, the combination of CV1 (56.2%) and CV2 (18.9%) separates

the three taxa. The cross-validation analysis of the CVA is presented in Table 2.2. All

second molars are correctly classified at the species level and only one Pt molar is

misclassified at the subspecies level. These results support our first hypothesis regarding

61

the taxonomic distinctiveness of EDJ shape for second molars. Contrary to the analysis of

first molars, the second hypothesis is supported as the distance in Procrustes shape space

is less between Ptt and Ptv than the distance between either subspecies and Pp (Table

2.4).

The mean shape of the second molar of each taxon is illustrated in Fig. 2.6. The

mean shape of Pp M2s compared to the mean shape of the combined Pt M2 sample is

visualized in Fig. 2.7a. The shape differences at the species level include relatively

centrally-placed dentine horns in Pt M2s compared to more laterally-placed dentine horns

in Pp M2s resulting in a relatively larger occlusal basin in the latter. The hypoconulid

dentine horn of Pt M2s is also relatively smaller compared to that of Pp M2s. The mean

shape differences in M2s of the two subspecies, Ptt and Ptv, are illustrated in Fig. 2.7b.

Ptt second molars exhibit a buccolingually-narrower crown base, relatively taller dentine

horns and a taller dentine crown, while Ptv molars exhibit a wider, more rectangular,

crown base, a wider occlusal basin, shorter dentine horns, and a shorter dentine crown.

Classification of specimens with unknown taxonomic affiliation

The specimens whose provenience, and thus taxonomy, is not certain (Table 2.3)

were included in a CVA analysis and classified in relation to the study sample. In some

cases we can make an informed guess regarding the taxonomy of these specimens based

on other information. We can also examine how stable the classification is for each

specimen within each of four analyses (Table 2.3). The basis for the classification of the

“unknown” first molars can be visualized in Fig. 2.2b and that of the “unknown” second

molars in Fig. 2.5b. These must be interpreted with caution as they only represent two

62

CV axes and thus they are susceptible to projection errors. A CVA, using the first 10 PCs

(83% of total variation) of a PCA based on the combined sample of first and second

molars, is illustrated in Fig. 2.8.

The three molars (from two individuals; #1, #9, #10) from the Royal Museum for

Central Africa were collected by the same individual and in the same year as other

specimens of Pp, and this is consistent with their classification as Pan paniscus in all of

the analyses. Specimen #8 and #13 are the first and second molars, respectively, from the

same individual; a captive specimen from a zoo in Berlin, Germany. The molars are

consistent in their classification of this individual as Ptv. Specimen #4 which, based on

the CVA analyses, is consistently classified as a first molar of P. paniscus. In the

museum catalogue this specimen is listed as being “taken on board in Matadi.” Matadi is

a city in Democratic Republic of the Congo located on the Congo River which divides

the ranges of Ptt and Pp. It is possible that this specimen belonged to a Pp individual, but

is incorrectly listed in the museum catalogue as belonging to Pt (it is also worth noting

that the specimen could have been catalogued prior to the recognition of Pp as a species).

Specimen #5, which was originally listed as a first molar is classified in this analysis as a

Ptt second molar. This specimen is listed as deriving from Katsema, Cameroon, which

would suggest that the taxonomic affiliation is correct. As this is the only specimen

misclassified to tooth type a return trip to the museum is planned to confirm that the tooth

type designation is sound. A number of specimens are consistent in their species and

subspecies designation (e.g., #1, #2, #4, #5, #8-13) while three are consistent in their

species designation but inconsistent in their subspecies classification (e.g., #3, #6, and

#7).

63

Discussion

The goal of this project is to assess the taxonomic distinctiveness of the EDJ

morphology of lower molars in species and subspecies of Pan. If it can be demonstrated

that the Pan EDJ carries information useful for distinguishing groups at these taxonomic

levels, then it is likely the same methods will have utility for discriminating among the

species and subspecies of other genera within the Pan/Homo clade. It is important to

establish reliable taxonomic hypotheses for fossil hominins, and to re-assess them as new

discoveries are made. Taxonomic decisions that partition the fossil record into the

hypodigms are essential for any further exploration of the evolutionary history and

paleobiology of the hominin clade.

There have been three previous studies of the taxonomic differences in tooth

morphology of Pan that are particularly relevant to the results of this study. The first was

by Johanson (1974) who examined metric and non-metric dental variation in Pp and in

the three subspecies of Pt and noted significant differences in both metric (MD and BL

dimensions) and non-metric variables between Pp and Pt and significant differences in

non-metric variables among the three subspecies of Pt. He also noted the distinctiveness

of Ptv compared to the other subspecies of Pt. Uchida (1992, 1996) examined

craniodental variation among hominoids. Her analyses of the dentition included linear

dimensions, cusp areas, and frequency of non-metric traits. Her findings echoed

Johanson’s regarding metrical differences in lower molar morphology between Pp and Pt,

but differed in that significant metrical differences were found between Ptv and Ptt

including: relative size of the protoconid on M1 and M2 (Ptv larger), relative size of the

64

hypoconid on M1 and M2 (Ptt larger), M2 crown shape (Ptv relatively wider

buccolingually). Cusp area measurements also distinguished Ptv from the other Pt

subspecies. The distinctiveness in EDJ morphology at the species and subspecies level

revealed in our analysis is consistent with these results.

Pilbrow (2003, 2006a) examined morphometric variation in African ape teeth,

including chimpanzee molars, based on a comprehensive set of linear measurements and

angles on the tooth crown. Pilbrow demonstrated significant morphometric variation

between species, subspecies and individual populations of Pan. Mahalanobis distances

between populations of chimpanzee resulted in four groups consistent with Pp, Ptv, Ptt,

and Pts. Based on shape variables, the classification accuracies in Pilbrow’s analysis

were: Pp – M1~70% and M2~88%; Pt – M1~90% and M2 ~93%; Ptt - M1 ~52% and M2

~62%; Ptv - M1~80% and M2~70%. Due to the small sample sizes of our analysis we do

not place strong confidence in the classification accuracies based on EDJ shape, however,

we believe it reasonable to conclude that at a minimum they are as good as, if not slightly

better than those based on the OES (particularly for the Ptt sample). We suggest that this

may be due to the fact that unlike methods that use 2D occlusal photographs our protocol

captures variation in crown height and dentine horn height.

The results we have presented indicate that EDJ morphology is distinctive at both

the species and subspecies level in Pan. Thus, the principles of parsimony would suggest

that the null hypothesis is that the EDJ morphology of the teeth of fossil hominins, who

share a most recent common ancestor with extant chimpanzees, is also likely to preserve

taxonomically relevant shape information at the same taxonomic levels. Indeed, EDJ

morphology, based on high-resolution CT images, is beginning to be used for the

65

diagnosis of new hominid taxa (Suwa et al., 2007). An analysis of EDJ shape in two

southern African hominin taxa, Australopithecus africanus and Paranthropus robustus,

indicates that EDJ morphology distinguishes both taxon and tooth type in lower molars

(Skinner et al., nd).

Genetic diversity is relatively low for Ptv and Pp compared to Ptt (Becquet et al.,

2007). While our results are consistent with this pattern with the Ptt samples covering a

larger proportion of shape space (and hence morphological variability) than Pp or Ptv,

this may also be due to sampling bias; particularly with the Ptv sample which derives

from a temporally and geographically restricted sample. Due to methodological

differences current molecular based estimates for the divergence time between Pan

species and subspecies differ among studies. Based on two recent analyses a cautious

estimate for the divergence of Pp and Pt is 1.0 – 1.5 Ma and between Ptv and the central

and eastern subspecies is 0.5 -0.9 Ma (Won and Hey, 2005; Becquet et al., 2007). Based

on the genetic data the predictions would be that the phenetic distance between Ptt and

Ptv would be less than the distance between either of the former taxa and Pp. The results

for the test of our second hypothesis, addressing whether EDJ morphology matches this

prediction are equivocal. The null hypothesis is rejected for first molars and accepted for

second molars. The small sample sizes for each group prevent a rigorous test of this

hypothesis and future research should increase sample sizes which would flesh out intra-

taxonomic patterns of variability.

Previous authors have suggested that the EDJ carries a more conservative

taxonomic signal than the OES (e.g., Korenhof, 1960; Sakai and Hanamura, 1973a,b;

Corruccini, 1987b; Sasaki and Hanazawa, 1999). While our results indicate that the EDJ

66

carries a strong taxonomic signal it is difficult to assess how this relates to the taxonomic

signal of the OES. This is for two reasons. First, unworn teeth are very difficult to find,

even in large museum collections, and second our methodology is difficult to apply to the

OES as the placement of landmarks on the more rounded surface of the OES is more

subjective than along the sharp ridge that runs between the dentine horns. Future research

into the taxonomic and phylogenetic utility of tooth structure (including EDJ and enamel

cap morphology) should increase sample sizes of high-resolution EDJ data to assess

expected levels of intraspecific variation. Furthermore, it may be that a combination of

EDJ morphology and the morphology of the enamel cap will be most effective for

distinguishing closely related taxa, as selection for OES crown morphology (or even the

functional morphology of a partially worn tooth, see below) can occur during the

formation of the EDJ and/or be related to the distribution of enamel deposited over the

EDJ.

As noted by Uchida (1996) it is difficult to point out any obvious functional

significance of the shape differences between subspecies and species. EDJ shape

differences are subtle and include slight variations in the shape of the crown base, the size

and height of the dentine horns and the placement of the dentine horns across the crown.

Such differences can result in increase in occlusal basin size and volume or the size of

shearing crests. However, the EDJ surface analyzed and visualized in this study does not

independently interact with food being masticated. In the unworn tooth that surface is the

culmination of EDJ shape and differential enamel distribution, and in partially worn teeth

can include the worn enamel cap and areas of exposed dentine which can create

functional crests in themselves (King et al., 2005). Enamel cap morphology and the

67

correlation between the EDJ and OES will need to be assessed in each taxon to determine

the extent to which EDJ shape is translated to the OES, and thus work out whether the

shape of the former can be used for interpreting the function of the latter.

Conclusion

This study tested the hypotheses that A) the EDJ morphology of molar tooth

crowns could distinguish between species and subspecies of extant chimpanzees, and B)

that the two subspecies of Pan troglodytes are more similar in shape to each other than

either is to Pan paniscus. The first hypothesis is supported based on the high accuracy

with which individual teeth are classified to the appropriate species and subspecies. The

second hypothesis was rejected for first molars and accepted for second molars and

requires further investigation. Morphological differences between extant Pan taxa are

subtle, but they can be visualized by the geometric morphometric approach employed in

this study. Our results suggest that EDJ morphology carries taxonomically relevant

information that can be incorporated into analyses of fossil hominids.

68

Table 2.1. First molars with their pre-established taxonomic affiliation and their

classification1 in each analysis.

Specimen Taxon M1_sp M1/M2_sp M1_ssp M1/M2_ssp

MRAC_27009_M1 Pan paniscus Pp Pp Pp Pp MRAC_29010_M1 Pan paniscus Pp Pp Pp Pp MRAC_29016_M1 Pan paniscus Pp Pp Pp Pp MRAC_29024_M1 Pan paniscus Pp Pp Pp Pp MRAC_29026_M1 Pan paniscus Pp Pp Pp Pp MRAC_29030_M1 Pan paniscus Pp Pp Pp Pp MRAC_29048_M1 Pan paniscus Pp Pp Pp Pp MRAC_5374_M1 Pan paniscus Pt Pp Ptv Ptt MPI_11791_M1 Pan t. verus Pt Pt Ptv Ptv MPI_11792_M1 Pan t. verus Pt Pt Ptv Ptv MPI_11798_M1 Pan t. verus Pt Pt Ptv Ptv MPI_11800_M1 Pan t. verus Pt Pt Ptv Ptv MPI_13433_M1 Pan t. verus Pt Pt Ptv Ptv MPI_14992_M1 Pan t. verus Pt Pt Ptt Ptt MPI_14995_M1 Pan t. verus Pt Pt Ptv Ptv MPI_15011_M1 Pan t. verus Pt Pt Ptv Ptv ZMB_0A16207_M1 Pan t. troglodytes Pt Pt Ptt Ptt ZMB_15849_M1 Pan t. troglodytes Pt Pt Ptt Ptt ZMB_30847_M1 Pan t. troglodytes Pp Pt Pp Ptt ZMB_35526_M1 Pan t. troglodytes Pt Pt Ptt Ptt ZMB_83604_M1 Pan t. troglodytes Pt Pt Ptt Ptt ZMB_83623_M1 Pan t. troglodytes Pp Pp_M2 Pp Pp_M2 ZMB_83673_M1 Pan t. troglodytes Pt Pt Ptt Ptt

Total 20/23 (87%)

22/23 (96%)

19/23 (83%)

20/23 (87%)

1. Misclassifications at the species level are shaded in dark grey and at the subspecies level in light grey. When tooth type is also misclassified the tooth type classification is listed.

69

Table 2.2. Second molars with their pre-established taxonomic affiliation and their

classification1 in each analysis.

Specimen Taxon M2_sp M1/M2_sp M2_ssp M1/M2_ssp

MRAC_22908_M2 Pan paniscus Pp Pp Pp Pp MRAC_29030_M2 Pan paniscus Pp Pp Pp Pp MRAC_29033_M2 Pan paniscus Pp Pp Pp Pp MRAC_29041_M2 Pan paniscus Pp Pp Pp Pp MRAC_29055_M2 Pan paniscus Pp Pp Pp Pp MRAC_29056_M2 Pan paniscus Pp Pp Pp Pp MRAC_29066_M2 Pan paniscus Pp Pp Pp Pp MRAC_84036M03_M2 Pan paniscus Pp Pp Pp Pp MRAC_84036M04_M2 Pan paniscus Pp Pp Pp Pp MPI_11790_M2 Pan t. verus Pt Pt Ptv Ptv MPI_11791_M2 Pan t. verus Pt Pt Ptv Ptv MPI_11792_M2 Pan t. verus Pt Pt Ptv Ptv MPI_11800_M2 Pan t. verus Pt Pt Ptv Ptv MPI_12176_M2 Pan t. verus Pt Pt Ptt Ptt MPI_13433_M2 Pan t. verus Pt Pt Ptv Ptv MPI_13437_M2 Pan t. verus Pt Pt Ptv Ptv MPI_14992_M2 Pan t. verus Pt Pt Ptv Ptv MPI_15008_M2 Pan t. verus Pt Pt Ptv Ptv MPI_15012_M2 Pan t. verus Pt Pt Ptv Ptv ZMB_30846_M2 Pan t. troglodytes Pt Pt Ptt Ptt ZMB_30847_M2 Pan t. troglodytes Pt Pt Ptt Ptt ZMB_31279_M2 Pan t. troglodytes Pt Pt Ptt Ptt ZMB_46095_M2 Pan t. troglodytes Pt Pt Ptt Ptt ZMB_83635_M2 Pan t. troglodytes Pt Pt Ptt Ptt ZMB_83639_M2 Pan t. troglodytes Pt Pt Ptt Ptt ZMB_83685_M2 Pan t. troglodytes Pt Pt Ptt Ptt

Total 26/26

(100%) 26/26

(100%) 25/26 (96%)

25/26 (96%)

1. Misclassifications at the species level are shaded in dark grey and at the subspecies level in light grey. When tooth type is also misclassified the tooth type classification is listed.

70

Table 2.3. List of specimens with unknown taxonomic affiliation and their classification in each analysis.

#1 Specimen Provenience information

2 M1_sp M1/M2_sp M1_ssp M1/M2_ssp

1 MRAC_84036M11_M1 Collected by the same individual as other Pp specimens (Pp?) Pp Pp Pp Pp 2 ZMB_0A809_M1 None (?) Pt Pt Ptv Ptv 3 ZMB_20811_M1 None (?) Pt Pt Ptv Ptt 4 ZMB_32052_M1 “Taken on board in Matadi” (?) Pp Pp Pp Pp 5 ZMB_32356_M1 Katsema, Cameroon (Ptt? Pt Pt Ptt Ptt_M2 6 ZMB_47506_M1 Zoo specimen from Berlin (?) Pt Pt Ptt Ptv 7 ZMB_6983_M1 Listed in museum catalogue as coming from west Africa (Ptv?) Pt Pt Ptv Ptt 8 ZMB_72844_M1 Zoo specimen from Berlin (?) Pt Pt Ptv Ptv Specimen Provenience information M2_sp M1/M2_sp M2_ssp M1/M2_ssp

9 MRAC_84036M10_M2 Collected by the same individual as other Pp specimens (Pp?) Pp Pp Pp Pp 10 MRAC_84036M11_M2 Collected by the same individual as other Pp specimens (Pp?) Pp Pp Pp Pp 11 ZMB_24838_M2 Bugoie Wald, Rwanda? (Pts) Pt Pt Ptt Ptt 12 ZMB_33489_M2 Egypt (?) Pt Pt Ptv Ptv 13 ZMB_72844_M2 Zoo specimen from Berlin (?) Pt Pt Ptv Ptv 1. Numbers at left indicate the location of specimens in the various PCA and CVA plots.

71

Table 2.4. Procrustes distance between each taxon for each analysis.

M1s (5PCs) PpM1 PttM1 PtvM1 Consistent with phylogenetic expectations?1

PpM1 0.000 PttM1 0.060 0.000 NO PtvM1 0.067 0.079 0.000

M2s (6PCs) PpM2 PttM2 PtvM2

PpM2 0.000 PttM2 0.076 0.000 YES PtvM2 0.075 0.070 0.000

M1/M2s (10PCs) PpM1/M2 PttM1/M2 PtvM1/M2

PpM1/M2 0.000 PttM1/M2 0.040 0.000 NO PtvM1/M2 0.042 0.046 0.000

1. To meet phylogenetic expectations the distance between Ptt and Ptv should be less than the distance of either subspecies to Pp.

72

Figure 2.1. EDJ surface model of a lower molar illustrating the anatomical landmarks used to capture EDJ shape. Landmarks are collected on the tips of the dentine horns and in the troughs between the mesial and buccal dentine horns (red spheres), along the RIDGE curve that runs between the dentine horns (purple spheres), and around the CERVIX curve (green spheres). Numbers refer to MAIN landmark set (see text for details).

73

A) B) Figure 2.2. A) PCA of the M1 sample. B) CVA of the M1 sample based on the first five PCs (explaining 72.3% of total variation). For both PCA and CVA grouping is by subspecies. Open stars with numbers indicate the position of specimens of unknown taxonomic affiliation (see Table 2.3). Abbreviations are as in text.

74

Figure 2.3. EDJ surface models of mean M1 shape in occlusal (top) and distal (bottom) view. A) P. paniscus; B) P. t. troglodytes; C) P. t. verus. Orange spheres denote anatomical landmarks used in shape analysis.

75

Figure 2.4. Comparison of mean EDJ shape of M1. A) Species-level comparison between the mean P. paniscus M1 (transparent) shape and the mean shape of the combined P.

troglodytes M1 (solid) sample. B) Subspecies-level comparison between the mean P. t.

troglodytes M1 (transparent) shape and the mean P. t. verus M1 (solid) shape.

76

A) B) Figure 2.5. A) PCA of the M2 sample. B) CVA of the M2 sample based on the first six PCs (explaining 75.1% of total variation). For both PCA and CVA grouping is by subspecies. Open stars with numbers indicate the position of specimens of unknown taxonomic affiliation (see Table 2.3). Abbreviations are as in text.

77

Figure 2.6. EDJ surface models of mean M2 shape in occlusal (top) and distal (bottom) view. A) P. paniscus; B) P. t. troglodytes; C) P. t. verus. Orange spheres denote anatomical landmarks used in shape analysis.

78

Figure 2.7. Comparison of mean EDJ shape of M2. A) Species-level comparison between the mean P. paniscus M2 (transparent) shape and the mean shape of the combined P.

troglodytes M2 (solid) sample. B) Subspecies-level comparison between the mean P. t.

troglodytes M2 (transparent) shape and the mean P. t. verus M2 (solid) shape.

79

Figure 2.8. CVA of the combined M1/M2 sample based on the first 10 PCs (explaining 83.0% of total variation). For CVA grouping is by subspecies and tooth type. Open stars with numbers indicate the position of specimens of unknown taxonomic affiliation (see Table 2.3). Abbreviations are as in text.

80

CHAPTER THREE: ENAMEL-DENTINE JUNCTION (EDJ) MORPHOLOGY

DISTINGUISHES THE LOWER MOLARS OF

AUSTRALOPITHECUS AFRICANUS AND PARANTHROPUS ROBUSTUS

Abstract

Tooth crown morphology plays a central role in hominin systematics, but the

removal of the outer enamel surface by dental attrition reduces, sometimes drastically, the

sizes of the samples available for study. This effectively eliminates from consideration

the type of detailed crown morphology that has been shown to discriminate among

hominin taxa. The enamel-dentine junction (EDJ) is the developmental precursor of the

outer enamel surface, yet its morphology is only affected after considerable attrition. In

this paper we explore whether the form of the EDJ can be used to distinguish between the

mandibular molars of two southern African fossil hominins: Paranthropus (or

Australopithecus) robustus (P. robustus) and Australopithecus africanus (A. africanus.)

We made high resolution images of the EDJ and used geometric morphometrics to

compare EDJ molar shape differences between species, in addition to documenting

metameric variation along the molar row within each species. Landmarks were collected

along the marginal ridge which runs between adjacent dentine horns and around the

circumference of the cervix. Our results suggest that the morphology of the EDJ can

distinguish lower molars of these southern African hominins, but it can also discriminate

first, second, and third molars within each taxon. These results confirm previous findings

that the EDJ is a useful taxonomic tool as it preserves taxonomically valuable shape

information in worn teeth.

81

Introduction

Given the prevalence of teeth in the hominin fossil record it is not surprising that

dental morphology plays a central role in hominin species diagnoses and that dental

evidence features prominently in attempts to generate phylogenetic hypotheses. Molar

crown morphology has been shown to be distinctive among extant higher primate species

and subspecies (Johanson, 1974; Uchida, 1992, 1996; Pilbrow, 2003, 2006), and between

many fossil hominin taxa (e.g., Weidenreich, 1937; Robinson, 1956; Sperber, 1974;

Wood and Abbott, 1983; Wood et al., 1983; Wood, 1991; Suwa et al., 1994; Suwa, 1996;

Bailey, 2002, 2006; Grine, 2004; Hlusko, 2004; Irish and Guatelli-Steinberg, 2003;

Guatelli-Steinberg and Irish, 2005). Unfortunately, dental attrition degrades the

information content of enamel morphology by removing measurable aspects of

homologous morphology. Using less comprehensive measures of tooth shape can result

in considerable overlap between taxa which can be well-differentiated based on other

skeletal anatomy.

It is known that the shape of the enamel-dentine junction (EDJ) quite closely

mirrors the shape of the outer enamel surface (OES) (Butler, 1956; Korenhof, 1960,

1961) and that unlike the OES the EDJ is preserved intact in partially worn teeth.

Previous analyses have demonstrated that primate EDJ morphology carries useful

taxonomic and phylogenetic information (e.g., Corruccini, 1987, 1998; Olejniczak et al.,

2004, 2007; Skinner et al., 2008) and can successfully discriminate between species and

subspecies of chimpanzee (Skinner, 2008). If the EDJ is at least as taxonomically

valuable as the OES then sample sizes of hominin teeth available for analysis could be

increased by adding teeth that have lost the OES either by antemortem attrition or by

postmortem abrasion, but which still retain the EDJ in its pristine state.

82

The morphology of lower molar tooth crowns, such as overall crown size, relative

cusp areas and molar size gradients, has been noted to distinguish P. robustus and A.

africanus (Robinson, 1956; Sperber, 1974; Wood and Abbott, 1983; Wood et al., 1983;

Suwa et al., 1994; Suwa, 1996; Grine, 2004). However, for many tooth crown variables

there is considerable overlap between these taxa. This study extends this research by

comparing both within- and between-species variation in EDJ morphology in A.

africanus and P. robustus. We do this by employing high resolution microCT to image

the EDJ surface, geometric morphometrics to capture the details of its shape, and

multivariate analytical methods to compare EDJ shape among taxa and molar position.

Materials and Methods

The study sample comprised isolated mandibular molars from collections housed

at the Transvaal Museum, Pretoria, South Africa and the University of Witwatersrand,

Johannesburg, South Africa (Table 3.1). Teeth from Swartktrans, Gondolin and Drimolen

(Robinson, 1956; Tobias et al., 1977; Grine 1989; Menter et al., 1999; Keyser et al.,

2000; De Ruiter, pers. comm. 2006) constitute the P. robustus sample, and teeth from

Sterkfontein Member 4 (Tobias et al., 1977; Moggi-Cecchi et al., 2006) make up the A.

africanus sample. These references were used to establish the taxonomy of each

specimen and in the case of isolated teeth, their position in the molar row. In some cases

molars are of known position because they derive from mandibular specimens (e.g., SK6)

or because they are part of an associated dentition (e.g., STW537). A sample of isolated

molars, whose position along the molar row is uncertain, is listed in Table 3.2 along with

previous classifications based on the above references (see also Suwa, 1996). These

83

molars are treated as unknown in our analysis and an attempt is made to assess their

likely position along the molar row using aspects of EDJ shape.

All molars preserved intact occlusal EDJ surfaces with the exception of three

specimens discussed below in the section describing EDJ surface reconstruction. Some

scanned teeth could not be included in this project because the effects of diagenesis were

such that it was not possible to tell the dentine from the enamel. In the case of teeth with

antimeres, the side that was better preserved tooth was included for analysis. Due to the

limitations of the object size that could fit into the portable microCT scanner used in this

study, few molars from mandibular specimens could be included.

Microcomputed tomography

Each tooth was microCT-scanned using a SKYSCAN 1172 Desktop Scanner

(100Kv, 94mA, 2.0mm aluminum and copper filter, 0.12 rotation step, 360 degrees of

rotation, 2 frame averaging). Raw projections were converted into TIFF image stacks

using NRecon (parameters: ring artifact correction = 10; beam hardening = 30%). Pixel

dimensions and slice spacing of the resultant images ranged between 10 – 20

micrometers, or microns (µm). To reduce the size of the resulting files, teeth were down-

sampled to 30 µm using Amira (Triangle filter).

EDJ surface reconstruction

To facilitate tissue segmentation, the complete image stack for each tooth was

filtered using a three-dimensional median filter (kernel size of 3) followed by a mean-of-

least-variance filter (kernel size of 3), implemented as a computer-programmed macro.

This filtering process results in more homogenous tissue classes (e.g., enamel vs. dentine)

84

and allocates pixels with intermediate gray-scale values at tissue interfaces (i.e., air-

enamel, enamel-dentine, air-dentine) to the appropriate tissue (Schulze and Pearce, 1994).

Filtered image stacks were imported into the Amira software package (v4.1,

www.amiravis.com) and enamel and dentine tissues were segmented using the 3D voxel

value histogram and its distribution of gray-scale values, which typically presents a

trimodal distribution with one peak representing dentine, another peak representing

enamel, and a third peak representing air and background noise in the images. In fossil

teeth where the enamel and dentine differ substantially in their degree of mineralization

(and thus in the ability of X-rays to pass through them), the filtering process results in

gray-scale pixel value distributions for each tissue that do not overlap. In other teeth,

diagenetic alteration (e.g., dentine remineralization) may result in similar tissue densities

and thus overlapping gray-scale pixel value ranges for enamel and dentine (Olejniczak

and Grine, 2006). Even after filtering, there is often an incomplete separation between the

two, and a decision must be made about the range of gray-scale values allocated to each

tissue. Only teeth in which there was a clear separation of enamel and dentine, resulting

in well-distinguished gray-scale values and accurate representations of the EDJ, were

used in the study.

After segmentation, the EDJ was reconstructed as triangle-based surface model

using Amira (surface generation module using unconstrained smoothing parameter).

Small portions of the EDJ are missing in some teeth, and in these cases the defects were

corrected digitally using the software Geomagic Studio 10. Teeth that showed evidence

of significant damage or missing areas were excluded from the study. SKX5002 and

GDA2 possess one (protoconid) and two (protoconid and hypoconid) exposed dentine

horn tips, respectively. In each case this exposure is minor and the tips were

85

reconstructed digitally (Fill holes option in Geomagic Studio 10). A crack was digitally

repaired in STW3 using surface editing functions in Geomagic Studio 10. In specimens

that preserved only the enamel cap, a surface model of the EDJ was created by digitally

removing the occlusal surface of the reconstructed enamel cap surface model.

Collection of landmarks

The EDJ surface models (PLY format) were imported into Amira 4.0 for the

collection of three sets of 3D anatomical landmarks (Fig. 3.1). The first set (referred to as

“MAIN”) included eight landmarks: one on the tip of the dentine horn of each primary

cusp [i.e., protoconid (1), metaconid (2), entoconid (3), hypoconid (4) and hypoconulid

(5)], one at the mid-point on the marginal crest connecting the protoconid and metaconid

(6), and one on the lowest point on the marginal ridge between the protoconid and

hypoconid (7) and the hypoconid and hypoconulid (8), respectively. Landmarks were not

collected in the trough between the metaconid and entoconid or between the entoconid

and hypoconulid because of the variable presence of accessory cusps in these areas which

violates the homology of these locations between molars.

The second set (referred to as the “RIDGE” curve) includes landmarks

(approximately 50-70) along the top of the ridges running between each of the five

dentine horns. This set of landmarks forms a closed ellipse, beginning at the tip of the

protoconid and moving in a lingual direction. In the case of teeth with accessory cusps

(e.g., cusp 6 or cusp 7) these dentine horns were ignored and points were collected on

either side.

The third set (referred to as the “CERVIX” curve) includes landmarks

(approximately 40-50) along the cervix, or cemento-enamel junction, of the tooth crown.

86

This set of landmarks also forms a closed ellipse, beginning below the protoconid dentine

horn and moving in a lingual direction. Where small fragments of enamel were missing at

the cervix its location was estimated.

Landmark datasets were exported as text files. Due to post-mortem damage the

CERVIX curve is not present in all specimens and thus sample sizes differ between

analyses. Specimens preserving both RIDGE and CERVIX curves (n = 23) and RIDGE

curves only (n = 28) are listed in Tables 3.1 and 3.2. We tested the influence of

downsampling (from 15-30 microns) and filtering (no filtering, kernel sizes of 1 and

kernel sizes of 3) on landmark configurations of a single specimen. The distance in

Procrustes shape-space between the original specimens and those that had been

downsampled or filtered with different settings were minimal and considerably less than

distances from the original specimen to any other individuals of the same species or tooth

type (Skinner, 2008).

Derivation of homologous landmarks

For each specimen a single set of homologous landmarks and semilandmarks was

derived from the three landmark files discussed above as follows. For both the RIDGE

and CERVIX curve landmark sets a smooth curve was interpolated using a cubic spline

function (a cubic spline is used so that the curve is forced to pass through each measured

coordinate). In the case of the CERVIX curve an arbitrary first point is chosen at the base

of the protoconid dentine horn and a cubic spline is fitted beginning at the first point and

moving lingually around the circumference of the cervix to the last point.

Interpolated curves were then resampled to achieve identical point counts between

specimens – these resampled points were used as semilandmarks. In the case of the

87

RIDGE curve, the eight MAIN landmarks were projected onto the curve dividing the

curve into eight sections. For each section a large sample of very closely spaced points

were computed along the curve and the distances between adjacent points were calculated

and summed together to approximate the length along the curve between MAIN

landmarks. Each length was divided by a given number and the coordinate location at

each equally spaced distance was recorded (the number of landmarks between MAIN

landmarks are illustrated in brackets in Fig. 3.1). In the case of the cervix curve, its length

was calculated in the same way and 70 equally spaced points were derived.

These equidistantly spaced points are then used as starting positions for the

semilandmark algorithm: semilandmarks were iteratively allowed to slide along their

respective curves (i.e., RIDGE curve [n =55] and CERVIX curve [n = 70]) to minimize

the bending energy of the thin-plate spline interpolation function computed between each

specimen and the Procrustes average for the sample. We used the algorithm of Bookstein

(1997; Gunz et al., 2005) that allows points to slide along tangents to the curve. These

tangents were approximated for each semilandmark by the vector between the two

neighboring points. Only the tips of the dentine horns were used as fixed landmarks, all

other points were treated as semilandmarks. After the application of the sliding algorithm

these semilandmarks were considered homologous for the purpose of multivariate

analyses.

Procrustes superimposition

The homologous landmarks and semilandmarks (after sliding) were converted to

shape coordinates by Generalized Least Squares Procrustes superimposition (Gower,

1975; Rohlf and Slice, 1990). This removed information about location and orientation

88

from the raw coordinates and standardized each specimen to unit centroid size, a size-

measure computed as the square root of the sum of squared Euclidean distances from

each landmark to the specimen’s centroid (Dryden and Mardia, 1998). All data

preprocessing was done in Mathematica v6.0 (www.wolfram.com) using a software

routine written by PG.

Analysis of EDJ shape

EDJ shape was compared in a number of different analyses within both shape

space (i.e., with all specimens size-standardized) and in form space (i.e., including the log

of centroid size as a variable along with the shape coordinates). By using both methods it

is possible to determine whether size and/or shape differentiates particular specimens.

Analyses were conducted using both CERVIX and RIDGE landmarks, CERVIX only,

and RIDGE only; for each of the foregoing combinations A. africanus and P. robustus

were analyzed as a single, combined, sample.

Two statistical methods were used to assess EDJ shape variation in the study

sample: principal components analysis (PCA) and canonical variates analysis (CVA). A

PCA of shape coordinates (Bookstein, 1991; Rohlf, 1993) after Procrustes

superimposition is an eigen-decomposition of the variance-covariance matrix of

Procrustes coordinates. A CVA was computed as linear discriminants in the software

package R (www.r-project.org) in the subspace of the first few principal components

(i.e., using only a subset of all PCs) of the Procrustes shape coordinates. It was necessary

to use a lower dimensional subspace because the number of variables greatly exceeded

our sample size. If too many variables are used to compute the discriminant axes it results

in unrealistic and unstable levels of discrimination. Our decision as to the number of PCs

89

to incorporate into the CVA was guided by a protocol in which the specimen labels were

randomized and the resulting CVA was assessed with variable numbers of PCs included.

The number of PCs included (in these analyses 5-8) did not exceed the point at which the

specimens with randomized labels began to exhibit clustering patterns in the resulting

CVA. In this way, we ensured that we only used a subset of PCs that did not generate

separation among the samples with randomly assigned group labels.

The accuracy with which EDJ shape correctly classifies molars, according to

species and molar position, was tested by cross validating the CVA. In this process each

specimen in turn was considered unclassified and then classified by all the other molars

of known position using posterior probabilities. Classification was implemented in R and

groups were assigned equal prior probabilities.

Visualization of EDJ shape variation

To visualize the shape variation between taxa and between molar position we

employed a method which allows a 3D triangulated surface reconstruction of the EDJ to

be deformed to match the mean configuration of particular molar positions for each taxon

(Gunz and Harvati, 2007). First, several thousand points were measured on the dentine

surface of one specimen (STW412B) and converted to a triangulated surface (PLY file

format) using Geomagic Studio 10. Because no shape data were collected within the

occlusal basin of the EDJ this area of the surface was purposely defeatured. We then used

the thin-plate spline interpolation function between the landmarks configuration of this

specimen and a template configuration in Procrustes space to warp the vertices of this

surface into Procrustes space. Finally we computed a thin-plate spline between this

template and a target form (e.g., the mean configuration of the P. robustus M1 sample) to

90

morph the vertices of the surface. In order to visualize the differences between the

surfaces representing the mean shapes of the molar representing each taxon, they were

superimposed in Amira with one surface rendered transparent for better visual

comparison.

Results

Figures 3.2a and 3.3a illustrate the results of the principal component analyses of

the complete landmark dataset (i.e., RIDGE curve and CERVIX curve) in shape space

and form space, respectively. While there is some separation between molar positions in

each plot the considerable degree of overlap on the first two PCs indicates broad size and

shape similarity among the molars in this sample. The canonical variate analysis of shape

space (using 7 PCs) and form space (using 8 PCs) of the complete landmark dataset are

illustrated in Figures 3.2b and 3.3b, respectively. Collectively, the first and second

canonical variates in each analysis separate taxa and molar position (the percentage of

total variance explained by each CV is listed). The combination of considerable overlap

along the first two principal component axes and separation in the CVA indicates that it

is small scale shape differences that separate taxa and molar position and the low

percentage of total variance explained by the first and second CVs in each analysis

indicates that these small scale shape differences account for relatively little of the overall

shape variation. Specimens of unknown molar position are plotted with stars in both the

PCA and CVA analyses. The reader is reminded that two dimensional plots of

multidimensional analyses can suffer from projection errors and therefore the spatial

association of unknown specimens with different groups should be not be over

interpreted. The PCA and CVA analyses of the RIDGE only and CERVIX only datasets

91

reveal similar results, albeit with somewhat less distinction in the CVA analyses (not

shown).

Classification using EDJ shape

Table 3.1 lists the classification to taxon and molar position of molars of known

position within each type of analysis (i.e., CERVIX and RIDGE, RIDGE only and

CERVIX only). Misclassifications to molar position are indicated with light shading and

to taxon with dark shading. The most reliable classification results from the inclusion of

shape information on both the RIDGE and CERVIX curves (78% and 91% in shape

space and form space respectively). The inclusion of centroid size as a variable (i.e., the

‘form’ columns in Table 3.1) also appears to increase the classification accuracy when

incorporating all landmarks, and for the RIDGE only analysis (79% and 93% in shape

space and form space, respectively). The CERVIX only analysis performs particularly

poorly with many misclassifications to both taxon and molar position (65% and 70% in

shape space and form space, respectively). The small sample sizes of all groups (but

particularly for A. africanus molars) render the results of the CVAs somewhat unstable.

Altering the number of PCs included in the CVA can result in different classifications

(usually to molar position) for one or two specimens, however, the relative degree of

separation between taxon and molar position and the overall classification accuracy does

not change substantially. The reasons for this instability are primarily the small sample

sizes for particular molar positions, but it may also be due to the similarity in shape

between certain molar types (see below).

The classification of unassigned molars is listed in Table 3.2. In addition to

classifications based on each curve, analyses were also conducted in shape space for each

92

taxon separately. As discussed above, the classification of these specimens is also

relatively unstable when differing numbers of PCs are included in the CVA, so we do not

consider these results reliable. One possible exception is SK104 which, in all analyses is

classified as a P. robustus first molar and this agrees with previous studies (Tobias et al.,

1977; Suwa, 1996).

Metameric molar shape variation

Figure 3.4 illustrates the mean shape of the first, second and third molars of A.

africanus and P. robustus. More direct comparisons of M1 vs. M2 and M2 vs. M3 shape

are illustrated in Figure 3.5 (A. africanus) and 6 (P. robustus). Some trends in shape

change between M1 and M3 are shared between these taxa. There is a reduction in crown

height between M1 and M2 (a pattern that is more marked in A. africanus, Fig. 3.5a) and

a marked reduction in the height of dentine horns between M1 and M2 (Figs. 3.5a/b,

3.6a/b). This pattern is not shared between M2 and M3, for the latter molar exhibits

relatively tall mesial dentine horns and a minor decrease in the relative height of the

distal dentine horns.

There is an increase in the mesiodistal length of the cervix along the molar row,

being shortest in M1 and longest in M3 (this difference is particularly marked in P.

robustus). There is a buccolingual expansion of the cervix in A. africanus M2 compared

to its M1, and a reduction in buccolingual breadth between M2 and M3 (particularly in

the distal half of the crown). A similar change in cervix shape is present in P. robustus,

with the reduction in the buccolingual breadth of the distal crown between M2 and M3

being even more marked, with the M3 having a more triangular crown base (Fig. 3.4f and

3.6d). In P. robustus, the marked inter-radicular extensions of the enamel cap that

93

characterize the M1 are less pronounced in the M2 (see also lingual view of M2 in Fig.

3.7c).

There are also changes in the relative position of the dentine horns that can be

assessed by comparing the position of their tips in the transparent and solid surface

models. Some noteworthy differences include the relatively more centrally placed dentine

horns on the talonid of the P. robustus M3 compared to the M2 (Fig. 3.6c/d). In contrast,

A. africanus has more centrally placed dentine horns on the M2 compared to the M3 (Fig.

3.5c/d).

Taxonomic differences in mean molar shape

Figure 7 illustrates comparisons between A. africanus and P. robustus at each

molar position. Dentine horn height is slightly higher in A. africanus compared to P.

robustus in all molar positions. Crown height is greater in the M1 of A. africanus

(ignoring somewhat the cervical extensions in the P. robustus M1). The dentine horns of

the A. africanus M2 appear more centrally placed (Fig. 3.7d) than in P. robustus (the

position of the dentine horns of the other molars does not substantially differ). The

lingual margin of the cervix of the A. africanus M2 is not centrally compressed as in P.

robustus and, not surprisingly, the distal half of the P. robustus M3 exhibits a

buccolingually narrow and distally extended cervix compared to that of A. africanus.

Discussion

Both A. africanus and P. robustus exhibit similar EDJ shape differences between

first, second and third molars, with some differences being more marked in one taxon

than in the other (e.g., distal expansion of the crown base in P. robustus M3s). It is a

94

reasonable, but untested assumption, that EDJ morphology, in conjunction with

differential enamel distribution, has an impact on tooth function and the metameric

differences in EDJ shape identified here imply different biomechanical responses of the

molars in each position under functional loading. Furthermore, these results suggest that

the exposure of dentine and the creation of secondary shearing crests (Shimizu, 2002;

King et al., 2005) would differ substantially between first molars and more distal molars

in both taxa. The taxonomic differences in EDJ shape, while consistently separating A.

africanus from P. robustus in discriminant analysis, are more subtle at each molar

position and this may result in equally subtle differences in functional morphology at

each position (i.e., A. africanus tending towards slightly taller dentine horns and crowns

which could be associated with greater shearing potential or resistance to effects of

abrasion). Thus, attempts to relate these morphological differences to differences in

molar function and inferred dietary adaptations between A. africanus and P. robustus

should take into account differences in the thickness and distribution of enamel over the

EDJ in these taxa.

The small-scale, but consistent shape differences in EDJ morphology present

between A. africanus and P. robustus, as well as between molar position within each of

these species, can be compared to previous analyses of crown morphology of the outer

enamel surface (e.g., Robinson, 1956; Wood and Abbott, 1983; Wood et al., 1983; Suwa

et al., 1994). The differences in relative dentine horn height seen in the mean EDJ shapes

are consistent with Robinson’s suggestion (1963; cited in Wood et al., 1983) that P.

robustus teeth are low cusped compared to A. africanus. But any differences are quite

small at the EDJ and the relative contributions of EDJ shape and enamel distribution

across the enamel cap to this pattern should be explored. Robinson (1956) claimed there

95

were differences in crown shape between A. africanus and P. robustus (e.g., oval in

Paranthropus and more rectangular in Australopithecus), but Wood and Abbott (1983)

could not find statistically significant differences in a measured index of crown shape. In

any event such differences are difficult to evaluate with our analysis of EDJ shape

because our assessment of crown shape comes primarily from the cervix while previous

analyses have examined dimensions of the enamel cap, which are influenced by

differential enamel deposition. However, examination of mean molar shapes does suggest

differences in the shape of the cervix between these taxa (particularly in the shape of P.

robustus M3s) even though such differences were less reliable in relation to

classification.

Wood and colleagues (1983) noted significant reduction the relative cusp areas of

the protoconid and metaconid (M1) and an increase in the areas of the hypoconulid (M1,

M2 and M3) in P. robustus compared to A. africanus. Similarly, Suwa and colleagues

(1994) found that P. robustus exhibits relatively smaller protoconid areas (M1, M2 and

M3), and relatively larger hypoconulid (M1) and entoconid (M2) areas. A number of

factors make it difficult to assess mean EDJ shape with respect to relative cusp size. The

locations of fissures, used to delineate cusps at the OES, are difficult to determine from

the EDJ as their pattern is dictated by a combination of EDJ shape, enamel extension

rates and the interaction of mineralizing fronts of ameloblasts at coalescence points.

Furthermore, the relative placement of dentine horns and the expansion or contraction of

the location of the cervix, as well as differential enamel distribution, all influence relative

cusp size as it is traditionally measured. Certainly the distal expansion of the cervix in P.

robustus agrees with the demonstrated increase in the relative size of the hypoconulid.

Comparisons of relative dentine horn volume could facilitate assessments of the relative

96

contribution of EDJ shape and enamel deposition to taxonomically distinctive patterns of

relative cusp size (Avishai et al., 2004).

Our results have demonstrated differences in lower molar shape between P.

robustus and A. africanus that have the potential to be incorporated into taxonomic and

tooth type assessments of isolated teeth. These findings are also consistent with Suwa

(1996) who used a combination of cusp area, crown shape and fissure pattern to achieve

accurate classifications of first and second molars within samples of A. africanus and P.

robustus, although classification accuracy was reduced when taxonomic affiliation was

treated as unknown. The higher accuracy of Suwa’s results compared to our own suggests

that patterns of enamel distribution between the two taxa may generate taxon specific

morphology that is not present at the EDJ (although the substantially larger samples used

by Suwa are also likely to be a contributing factor). Assessing reliability of hominin EDJ

shape for these purposes will require larger samples and in particular, molars from jaws

whose position is certain and whose taxonomic affiliation can be based on other aspects

of craniodental morphology. Currently, we have no similar data for East African fossil

hominins such as P. boisei and P. aethiopicus, which would allow an assessment of the

implications for the shape differences identified here to the ongoing debates regarding the

monophyletic nature of Paranthropus (see Wood and Constantino, 2007). The debate

about Paranthropus monophyly would also benefit from equivalent analyses of EDJ

shape variation in upper molars and upper and lower premolars.

Conclusion

The results of this study show that there are small-scale, but consistent differences

in EDJ shape between the lower molars of A. africanus and P. robustus and that these

97

taxa share consistent patterns of EDJ shape that serve to distinguish first, second, and

third molars. The EDJ preserves much of the shape information that is preserved on the

outer enamel surface of unworn and partially worn teeth and can be used to incorporate

worn teeth into taxonomic and phylogenetic analyses. This will result in larger samples

for nearly all early hominin taxa. Furthermore, EDJ shape may be useful in assessing the

taxonomic affiliation and tooth type (if it is ambiguous) of unknown specimens, as well

as allowing researchers to reassess previous assignments. The reliability of EDJ shape to

classify molars would benefit from the inclusion of specimens from mandibles whose

molar position is certain and whose taxonomic affiliation can be based on other aspects of

morphology.

98

Table 3.1. Study sample and classification accuracy of molars whose position is considered known.

Specimen Molar position RIDGE and CERVIX –

Shape (7PCs)

RIDGE and CERVIX –

Form (8PCs)

CERVIX ONLY - Shape

(8PCs)

CERVIX ONLY –

Form (8PCs)

RIDGE ONLY –

Shape (8PCs)

RIDGE ONLY –

Form (8PCs) DNH60B P. robustus M1 P. rob M2 P. rob M1 P. rob M1 P. rob M1 P. rob M1 P. rob M1 SK828 P. robustus M1 P. rob M1 P. rob M1 P. rob M1 P. rob M1 P. rob M1 P. rob M1 SK843 P. robustus M1 P. rob M1 P. rob M1 P. rob M1 P. rob M1 P. rob M1 P. rob M1 STW309A A. africanus M1 A. afr M1 A. afr M1 A. afr M2 A. afr M2 A. afr M1 A. afr M1 STW421A A. africanus M1 A. afr M1 A. afr M1 A. afr M2 P. rob M2 P. rob M1 P. rob M1 DNH60C P. robustus M2 P. rob M2 P. rob M2 P. rob M2 A. afr M3 P. rob M2 P. rob M2 SK6 P. robustus M2 P. rob M2 P. rob M2 P. rob M2 P. rob M2 P. rob M2 P. rob M2 SK843 P. robustus M2 P. rob M2 P. rob M2 P. rob M2 A. afr M2 P. rob M2 P. rob M2 SK1587B P. robustus M2 P. rob M2 P. rob M2 P. rob M2 P. rob M2 P. rob M2 P. rob M2 STW213 A. africanus M2 A. afr M2 A. afr M2 A. afr M2 P. rob M2 A. afr M2 A. afr M2 STW308 A. africanus M2 ---- ---- ---- ---- A. afr M2 A. afr M2 STW412B A. africanus M2 A. afr M2 A. afr M2 A. afr M3 A. afr M2 A. afr M2 A. afr M2 STW424 A. africanus M2 ---- ---- ---- ---- A. afr M2 A. afr M2 STW537 A. africanus M2 A. afr M3 A. afr M2 A. afr M2 A. afr M2 A. afr M2 A. afr M2 STW560E A. africanus M2 A. afr M2 A. afr M2 A. afr M3 A. afr M3 A. afr M2 A. afr M2

SK6 P. robustus M3 P. rob M3 P. rob M3 A. afr M3 A. afr M3 P. rob M3 P. rob M3 SK22 P. robustus M3 P. rob M3 P. rob M3 P. rob M3 P. rob M3 P. rob M3 P. rob M3 SK75 P. robustus M3 P. rob M3 P. rob M3 P. rob M3 P. rob M3 A. afr M3 P. rob M3 SK841B P. robustus M3 P. rob M3 P. rob M3 P. rob M3 P. rob M3 A. afr M3 A. afr M3 SK851 P. robustus M3 ---- ---- ---- ---- P. rob M3 P. rob M3 SKX5002 P. robustus M3 P. rob M3 P. rob M3 P. rob M2 P. rob M3 P. rob M3 P. rob M3 SKX5014 P. robustus M3 A. afr M3 A. afr M3 P. rob M3 P. rob M3 A. afr M3 P. rob M3 SKX10642 P. robustus M3 P. rob M2 P. rob M3 P. rob M3 P. rob M3 P. rob M3 P. rob M3 STW280 A. africanus M3 ---- ---- ---- ---- A. afr M3 A. afr M3 STW491 A. africanus M3 P. rob M3 A. afr M3 P. rob M3 P. rob M3 A. afr M3 A. afr M3 STW529 A. africanus M3 A. afr M3 A. afr M3 A. afr M2 A. afr M2 P. rob M3 A. afr M3 STW537 A. africanus M3 A. afr M2 A. afr M2 A. afr M2 A. afr M2 A. afr M2 A. afr M3 STW560B A. africanus M3 ---- ---- ---- ---- A. afr M3 A. afr M3

99

Table 3.2. Classification of unassigned molars within each analysis.

Specimen Previous molar position assignments1

RIDGE and CERVIX –

Shape (7PCs)

RIDGE and CERVIX –

Form (8PCs)

CERVIX ONLY –

Shape (8PCs)

CERVIX ONLY –

Form (8PCs)

RIDGE ONLY –

Shape (8PCs)

RIDGE ONLY –

Form (8PCs)

P. robustus

RIDGE and CERVIX –

Shape (7PCs)

A. africanus

RIDGE and CERVIX –

Shape (5PCs) P. robustus DNH67 M1 (4) ---- ---- ---- ---- P. rob M1 P. rob M1 ---- ---- GDA2 M2 (3) P. rob M2 P. rob M3 P. rob M2 A. afr M2 P. rob M3 P. rob M3 P. rob M2 ---- SK1 M2 (1) P. rob M1 P. rob M2 P. rob M1 P. rob M2 P. rob M2 P. rob M2 P. rob M1 ---- SK104 M1 (1); M1 (2) P. rob M1 P. rob M1 P. rob M1 P. rob M1 P. rob M1 P. rob M1 P. rob M1 ---- SK3974 M1 (1) P. rob M1 P. rob M1 P. rob M2 P. rob M1 P. rob M2 P. rob M2 P. rob M2 ----

A. africanus

STS9 M1 (1); M1/2 (2) P. rob M2 P. rob M2 A. afr M2 A. afr M2 A. afr M3 P. rob M2 ---- A. afr M3 STW3 M2 (5) A. afr M2 A. afr M3 A. afr M2 A. afr M2 A. afr M3 A. afr M3 ---- A. afr M2 STW145 M1 (5) A. afr M2 A. afr M2 A. afr M1 A. afr M1 A. afr M2 A. afr M2 ---- A. afr M2 STW520 M3 (5) ---- ---- ---- ---- A. afr M2 A. afr M2 ---- ---- 1. Molar position assignments based on the following references: 1) Tobias et al., 1977; 2) Suwa, 1996; 3) Menter et al., 1999; 4) Keyser et al., 2000; 5) Moggi-Cecchi et al., 2006

100

Figure 3.1. EDJ surface model of a lower molar (SK1587B) illustrating the anatomical landmarks used to capture EDJ shape. MAIN landmarks are collected on the tips of the dentine horns and in the troughs between the mesial and buccal dentine horns (red spheres), along the RIDGE curve that runs between the dentine horns (yellow spheres), and around the CERVIX curve (green spheres). Numbers in brackets refer to the number of equally spaced semilandmarks derived between MAIN landmarks for the RIDGE curve and on the CERVIX curve (see text for details). Landmarks shown here are those collected on the original specimens and are not the same as those generated from the sliding semi-landmark routine used to create a homologous set of landmarks between all specimens. Note that landmarks are collected on either side of the C6 dentine horn and are not included in the RIDGE curve.

101

A) B) Figure 3.2. PCA (A) and CVA (B) of EDJ morphology in shape space. CVA calculated using the first seven principal components separates both taxon and molar type. Groups are identified using polygons, while the two A. africanus M1s are joined by a line. Specimens whose molar type is uncertain are denoted by stars. Spatial association between specimens should be interpreted with caution as only two dimensions of a multidimensional shape space are represented.

102

A) B) Figure 3.3. PCA (A) and CVA (B) of EDJ morphology in form space. CVA calculated using the first eight principal components separates both taxon and molar type. Groups are identified using polygons, while the two A. africanus M1s are joined by a line. Specimens whose molar position is uncertain are denoted by stars. Spatial association between specimens should be interpreted with caution as only two dimensions of a multidimensional shape space are represented.

103

Figure 3.4. Illustrations of the mean shape of each molar type for A. africanus and P.

robustus. These surface models are generated by warping a generic EDJ surface to match the mean configuration of RIDGE and CERVIX landmarks for each group. Note the differences in relative dentine horn height and cervix shape.

104

Figure 3.5. Comparison of metameric variation along the molar row in A. africanus. In each case one surface is made transparent to facilitate comparison. M1 compared to M2 in buccal (A) and distal (B) view. M2 compared to M3 in buccal (C) and distal (D) view.

105

Figure 3.6. Comparison of metameric variation along the molar row in P. robustus. In each case one surface is made transparent to facilitate comparison. M1 compared to M2 in buccal (A) and distal (B) view. M2 compared to M3 in buccal (C) and distal (D) view.

106

Figure 3.7. Taxonomic differences in EDJ shape at each molar position. In each case one surface is made transparent to facilitate comparison. First molar comparison in lingual (A) and distal (B) view. Second molar comparison in lingual (C) and distal (D) view. Third molar comparison in lingual (E) and distal (F) view.

107

CHAPTER 4: DENTAL TRAIT EXPRESSION AT THE ENAMEL-DENTINE

JUNCTION OF LOWER MOLARS IN EXTANT AND FOSSIL HOMINOIDS

Abstract

Discrete dental traits are used as proxies for biological relatedness among modern

human populations and for alpha taxonomy and phylogeny reconstruction within the

hominin clade. We present a comparison of the expression of lower molar dental traits

(cusp 6, cusp 7, trigonid crest pattern, and protostylid) at the enamel-dentine junction

(EDJ) in a variety of extant and fossil hominoid taxa, in order to assess the contribution

of the EDJ to the morphology of these traits at the outer enamel surface (OES). Molars (n

= 44) were imaged nondestructively using high-resolution microCT, and three-

dimensional surface models of the EDJ and OES were created to compare trait expression

at each surface. Our results indicate that these dental traits originate at the EDJ, and that

the EDJ is primarily responsible for their degree of expression at the OES. Importantly,

variable trait morphology at the EDJ (often not easily recognizable at the OES) indicates

that different developmental processes can produce traits that appear similar at the

enamel surface, suggesting caution in intra- and intertaxonomic comparisons. The results

also highlight the importance of the EDJ for understanding the morphological

development of discrete traits, and for establishing graded scales of variation to compare

trait frequency among groups for the purpose of taxonomic and/or phylogenetic analysis.

Finally, this study demonstrates that imaging the EDJ of both worn and unworn fossil

hominin teeth provides a novel source of information about tooth development and

variation in crown morphology.

108

Introduction

Tooth crown morphology plays a critical role in hominin systematics in that it is

relevant to hypotheses of alpha taxonomy, the assignment of fossil specimens to hominin

taxa, and the reconstruction of the evolutionary history of the hominin clade. The

presence and degree of expression of discrete traits at the outer enamel surface (OES) of

teeth is an important component of these morphological analyses. Over the last three

decades, a concerted effort has been made to standardize the classification of dental traits

and to investigate how their expression varies within and among modern human

populations (e.g., Turner et al., 1991). Discrete dental traits have come to play a central

role in inferring biological relationships among modern humans (Scott and Turner, 1997,

and references therein), living nonhuman primates (Johanson, 1974; Uchida, 1996;

Pilbrow, 2003, 2006a), fossil hominoids (Pilbrow, 2006b), and fossil hominins (e.g.,

Weidenreich, 1937; Robinson, 1956; Wood and Abbott, 1983; Suwa et al., 1996; Bailey,

2002; Hlusko, 2004; Bailey and Lynch, 2005; Guatelli-Steinberg and Irish, 2005; Bailey

and Wood, 2007; Martinón-Torres et al., 2007).

Traditional analyses of discrete traits make two assumptions. First, trait

morphology that appears similar at the OES in different teeth is the result of

developmental processes that are similar enough to allow valid comparisons within and

between groups. Second, the OES is adequate for the formulation of standardized

classifications of trait variation (often taking the form of grades from minor to marked

trait expression). However, given how teeth grow, it is possible that different

developmental processes can result in similar morphology at the OES, confounding the

definition, classification, and comparison of discrete-trait morphology. During molar

tooth development, major aspects of crown morphology such as cusps form on a

109

basement membrane (membrana praeformativa) that serves as the template upon which a

layer of enamel is deposited (Butler, 1956, 1999). In mature teeth, the shape of this

membrane is preserved as the enamel-dentine junction (EDJ), and trait expression at the

OES is the culmination of EDJ shape and differential enamel distribution. This study

tests these assumptions about trait morphology at the OES by examining the shape of the

enamel-dentine junction (EDJ) as a proxy of trait development that is preserved in fully

formed teeth.

To understand how different developmental processes can result in similar trait

morphology at the OES, consider the following analogy. An artist is producing two

sculptures (equivalent to the fully formed tooth crown) using a wireframe template

(equivalent to the EDJ) and clay (equivalent to enamel). The artist can produce two

sculptures that are identical at their outer surfaces in two ways. First he can begin with

identically shaped wireframes and place an identical distribution of clay over each.

Conversely, he can begin with wireframes that differ in shape but apply different

distributions of clay over each to produce an identical shape at the surface. Furthermore,

two sculptures that differ in the shape of their surfaces can each contain identical

wireframes. Few studies of discrete dental traits have included information about the

shape of the wireframe, or EDJ, template (but see Schwartz et al., 1998).

The utility of the EDJ for understanding the developmental basis of crown

morphology has been demonstrated by a number of previous studies (Korenhof, 1960,

1961, 1982; Nager, 1960; Kraus, 1952; Kraus and Jordan, 1965; Sakai et al., 1965,

1967a,b, 1969; Sakai and Hanumura, 1971, 1973a,b; Corruccini 1987a,b, 1998, Schwartz

et al., 1998; Sasaki and Kanazawa, 1999; Olejniczak et al., 2004; Macchiarelli et al.,

2006). Most of these studies showed that the gross morphology of the OES is primarily

110

determined by the shape of the EDJ, with the shape of the enamel cap having only a

minor influence on the morphology of the OES. Butler (1956: 32–33) noted: “Allowing

for such modifications due to the deposition of enamel it remains true that the main

features of the crown pattern, and many of its minor details, are already present in the

membrane praeformativa before the hard tissues have developed.” However, the relative

contribution of the EDJ and the enamel cap to the expression of the smaller

morphological features of the tooth crown, which constitute discrete traits of fossil and

living hominins and hominoids, remains a topic of debate. Depending on the feature in

question, both concordance and a lack of concordance between the EDJ surface and OES

have been reported (e.g., Korenhof, 1960; Kraus, 1952; Corruccini and Holt, 1989;

Schwartz et al., 1998; Olejniczak et al., 2004).

Nager (1960) decalcified 96 human teeth to compare the shapes of the OES and

the EDJ of the same tooth. Based on his observations he defined three types of structures.

(Nager [1960] used the term crown relief to refer to morphological structures, but for the

purpose of this discussion, we will use the term trait.) A “primary-definitive” trait

consists of structures that are present on both the EDJ and on the unworn OES. This

category includes structures whose morphology is altered slightly when enamel is

deposited over the surface of the growing tooth (e.g., the discrepancy between a pointed

dentine horn and its overlying, more blunt enamel cusp does not preclude the “cusp”

from being a primary-definitive trait). A “primary-temporary” trait consists of structures

that are present on the EDJ, but cannot be observed on the unworn OES. An example of

the latter is the hypocone-protocone ridge present at the EDJ of human upper molars,

which is not visible at the enamel surface (Korenhof, 1960: Plate XIII, specimen MMSD

381). A “secondary” trait consists of structures not seen on the EDJ, but which are

111

evident on the OES (e.g., primary occlusal fissures present at the OES that have no

corresponding fissurelike morphology at the dentine surface).

This study examines four dental traits of the lower molarscusp 6, cusp 7,

trigonid crest pattern, and the protostylidwith the aim of determining the relative

contributions of the EDJ and the enamel cap to their expression at the OES in a variety of

extant and extinct hominoids (Fig. 4.1). These traits are found in all hominin and extant

great ape species, and they are thought to provide information about taxonomy and/or

phylogeny (e.g., Johanson, 1974; Wood and Abbott, 1983; Suwa et al., 1996; Uchida,

1996; Pilbrow, 2003, 2006b; Bailey, 2002, 2006; Bailey and Hublin, 2006; Bailey and

Wood, 2007; Martinón-Torres et al., 2007). A cusp 6 (also called a tuberculum sextum or

entoconulid, and referred to hereafter as C6) is a cusp or cuspule on a lower molar within

the distal fovea, lingual to the hypoconulid, or cusp 5 (Turner et al., 1991). A cusp 7 (also

called a tuberculum intermedium, interconulid, or metaconulid, and referred to hereafter

as C7) is a cusp or cuspule occurring in the lingual groove between the metaconid and

entoconid (Turner et al., 1991; Hlusko, 2002). Trigonid crest pattern refers to the

midtrigonid crest (defined as a transverse ridge or loph that connects the middle part of

the two mesial cusps) and the distal trigonid crest (defined as a transverse ridge or loph

that connects the distal aspect of the two mesial cusps) (Korenhof, 1982; Wu and Turner,

1993). A protostylid was described by Dahlberg (1950: 16) as “an elevation or ridge of

enamel on the anterior part of the buccal surface of the lower molars, which ascends from

the gingival end of the buccal groove and extends mesio-occlusally.”

We ask three questions about each trait. Does it originate at the EDJ? What is the

relative contribution of the EDJ to trait expression at the OES? Is the process of trait

development, as inferred from the shape of the EDJ and overlying enamel cap, consistent

112

among the study taxa? If a trait can be considered primary-definitive under Nager’s

classification, then the EDJ expression of a trait can be incorporated into its formal

definition and may inform the establishment of taxon-specific trait-scoring standards; the

importance of the latter has been noted by a number of authors (e.g., Reid and Van

Reenen, 1995; Van Reenen and Reid, 1995; Irish and Guatelli-Steinberg, 2003; Hlusko,

2004; Bailey and Wood, 2007). Furthermore, if it is shown that the EDJ is either a proxy

for OES morphology or is more informative than the OES, then worn fossil teeth may be

used in the analysis of discrete traits.

Materials and Methods

Study sample

Table 4.1 lists the molars included in this study. A range of living and extinct

hominid and hominin taxa (following the taxonomy of Wood and Richmond, 2000) were

included to capture variation for each trait within taxa, as well as variation in trait

morphology among taxa. While sex is known for some specimens, it is not incorporated

as a variable in our analysis due to the limited sample sizes for all taxa. First, second, and

third molars were included, and in a few cases, metameric teeth from the same individual

were examined. Modern human specimens include North American aboriginals and Early

Bronze Age specimens from Great Britain. Extant hominoids include Pan troglodytes

verus, Gorilla gorilla beringei, and Pongo pygmaeus ssp. The fossil taxa include

Gigantopithecus blacki, Australopithecus africanus, Paranthropus robustus, and Homo

neanderthalensis.

Micro-computed tomography and surface reconstruction

113

In order to produce three-dimensional reconstructions of the EDJ and the OES,

each tooth was scanned using either the SKYSCAN 1172 Desktop MicroCT (scan

parameters: 100Kv, 94µA, 2.0mm aluminum and copper filter, 2048 x 2048 matrix, 0.12

rotation step, 360 degrees of rotation, 2 frame averaging) or SCANCO µCT40 (scan

parameters: 70Kv, 114µA, 1024 x 1024 matrix, 0.36 rotation step, 180 degrees of

rotation) computed tomographic scanner. Pixel dimensions and slice thickness between

reconstructed serial images were isometric with resolutions ranging between 13 and 50

microns (µm) (e.g., isometric voxels of 13 µm × 13 µm × 13 µm to 50 µm × 50 µm × 50

µm).

To facilitate tissue segmentation, the complete image stack for each tooth was

filtered using a three-dimensional median filter (kernel size of 3), followed by a mean of

least variance filter (kernel size of 3), implemented as a computer-programmed macro.

This filtering process results in more homogeneous tissue classes (e.g., enamel vs.

dentine) and allocates pixels with intermediate gray-scale values at tissue interfaces (i.e.,

air-enamel, enamel-dentine, air-dentine) to the appropriate tissue (Schulze and Pearce,

1994). The effect of the filtering process on the morphology of the reconstructed surfaces

was assessed by overlaying surfaces derived from unfiltered images over surfaces derived

from filtered images and examining differences in shape. The effect of filtering on

surface morphology is minimal compared to the size of the structures (e.g., cusps and

crests) that constitute the discrete traits being analyzed. An exception is that the size and

shape of very small tubercles (e.g., <0.5 mm in length) cannot be discerned using this

methodology (discussed below).

Filtered image stacks were imported into the Amira software package (v4.1,

www.amiravis.com), and enamel and dentine tissues were segmented by evaluating the

114

3D-voxel-value histogram and its distribution of gray-scale values, which typically

presents a trimodal distribution with one peak representing dentine, another peak

representing enamel, and a third peak representing air and background noise in the

images. For unfossilized teeth of extant taxa, in which enamel and dentine tissues differ

substantially in their degree of mineralization (and therefore their densities and the ability

of X-rays to pass through them), the filtering process results in gray-scale pixel-value

distributions for each tissue that do not overlap. In fossil teeth, diagenetic alteration (e.g.,

dentine remineralization) sometimes results in similar tissue densities for enamel and

dentine, and thus overlapping gray-scale pixel-value ranges for each tissue (Olejniczak

and Grine, 2006). Even after filtering, there is often an incomplete separation between the

two, and a decision must be made about the range of gray-scale values allocated to each

tissue. All of the teeth in this study evinced a clear separation of enamel and dentine,

resulting in well-distinguished gray-scale values and accurate representations of the EDJ.

Specimens that could not be segmented to produce accurate surface reconstructions were

excluded from the study.

After segmentation, the OES and the EDJ were reconstructed as triangle-based

surface models in Amira 4.0 (surface generation module using unconstrained smoothing

parameter), which can be rotated and enlarged interactively to view and compare trait

expression. In specimens that preserved only the enamel cap, a surface model of the EDJ

was created by digitally removing the occlusal surface of the model of the reconstructed

enamel cap surface.

115

Results

The manifestation of each trait at the EDJ and the influence of enamel deposition

on trait manifestation at the OES are described below. Trait distribution within and

among taxa is addressed, although sample sizes are too small to warrant strong inferences

to be drawn regarding intertaxonomic differences in trait presence/expression.

Cusp 6

Variation in C6 manifestation at the EDJ of molars in the study sample is

summarized schematically in Figure 4.2 and can be separated into two types. The first

type, referred to here as the “hypoconulid-type” C6, is characterized by a dentine horn

(DH) on the lingual slope of the hypoconulid DH (Figs. 4.2b, 4.3a). The shape of this

feature resembles a smaller version of the adjacent hypoconulid DH. In a number of

specimens, this DH was duplicated, with both horns occurring in proximity to the

hypoconulid DH and resembling serially developed structures. These specimens are

characterized as having a double hypoconulid-type C6 (Figs. 4.2c, 4.3b). These types of

C6 are most common in the P. t. verus sample but were also present in the H. sapiens

sample (Table 4.2). The close association of this type of C6 with the hypoconulid is not

always apparent from the OES, particularly in partially worn teeth. Furthermore, in some

chimpanzee teeth, a double hypoconulid-type C6 can only be identified at the EDJ (Fig.

4.3b) due to tooth wear on the distal margin of the tooth crown.

The second type of C6, referred to here as the “fovea-type” C6, takes the form of

a DH on the marginal ridge of the distal fovea between the hypoconulid and entoconid

(Figs. 4.2d, 4.3c). This type can be differentiated from the hypoconulid-type because its

DH is separate from the hypoconulid DH and entoconid DH. In high-cusped teeth, such

116

as those of G. g. beringei, this type of C6 can be a tubercle on a cingulumlike shelf on the

distal marginal ridge of the EDJ (seen on the OES of Fig. 4.1). Taxa exhibiting the fovea-

type C6 include G. blacki, A. africanus, P. robustus, H. neanderthalensis, P. pygmaeus

ssp., and G. g. beringei. Similar to the hypoconulid-type discussed above, the fovea-type

C6 can also appear in a duplex form, with two DH-like features on the margin of the

distal fovea (schematically represented in Fig. 4.2e). One specimen of H. sapiens had

both types of C6 at the EDJ (schematically represented in Fig. 4.2f).

In both A. africanus and P. robustus, which have the fovea-type C6 when present,

enamel distribution has a marked influence on the shape of the C6 at the OES. The small

DH(s) at the EDJ corresponds with a relatively large cusp at the OES. This contrasts with

all of the other taxa, in which the relative size of the C6 DH(s) is similar to the relative

size of the C6 cusps at the OES. Thus, in these thickly enameled taxa (Grine and Martin,

1988), enamel distribution has a greater influence on C6 shape at the OES than in thinly

enameled taxa.

Another variant (possibly similar developmentally to a C6) was observed in one

P. t. verus molar and in one G. g. beringei molar (Fig. 4.3d). In these specimens, a small

cusp is present on the OES on a crest that joins the entoconid to the hypoconulid (the

presence of this crest is variable within and among most of the study taxa). In both teeth,

this cusp corresponds to a DH at the EDJ located on a crest between the entoconid DH

and hypoconulid DH.

Cusp 7

Variation in C7 manifestation at the EDJ of molars in the study sample is

summarized schematically in Figure 4.4. Like the C6 trait, there are two main types of

117

C7. The first type of C7, referred to as the “metaconulid-type,” is manifested at the EDJ

as a protuberance or DH-like feature on the end of a shoulder on the distal ridge of the

metaconid DH (Fig. 4.4c and 4.5a). This shoulder can be faint or pronounced, and it can

occur at varying distances from the metaconid DH (Figs. 4.4d, 4.5b). This type seems

consistent with grade 1A of the ASUDAS classification of cusp 7 (Turner et al., 1991).

The second type of C7, referred to as the “interconulid-type,” is manifest at the

EDJ by a DH of variable size on the low point of the marginal ridge between the DHs of

the metaconid and entoconid (Figs. 4.4f, 4.5c,d). In some cases, this type of C7 appears

more spatially associated with the metaconid, as evidenced by a less-developed trough

between the C7 DH and the metaconid compared to the trough between it and the

entoconid DH (Fig. 4.4e). This type corresponds with grades 1–4 of the ASUDAS

classification of cusp 7. The metaconulid-type C7 is present in P. t. verus, G. g. beringei

and A. africanus, while the interconulid-type is present in H. sapiens, H.

neanderthalensis, P. robustus, A. africanus, and G. blacki; no C7 was observed in P.

pygmaeus.

Differential enamel distribution does not greatly influence the manifestation of C7

at the OES, but distinguishing between a cuspule at the OES over a pronounced shoulder

of the metaconid, and a cuspule at the OES over a small DH on the shoulder of the

metaconid, is difficult based on OES morphology alone. Dental attrition does not obscure

C7 morphology at the OES in this sample. In one A. africanus (STW 560A) specimen,

the OES exhibits both of the types of C7 discussed here (the EDJ presenting a DH under

the interconulid-type and only a slight ridge elevation below the metaconulid-type C7).

Trigonid crest pattern (TCP)

118

Variation in trigonid crest patterning at the EDJ is summarized schematically in

Figure 4.6. The first type consists of either weakly pronounced or well-pronounced crests

on the slopes of the protoconid and metaconid DHs, which extend from the tips of the

DHs towards the occlusal basin (Figs. 4.6a,b and 4.7a). The second type consists of

pronounced but separate crests that extend across the occlusal basin (Figs. 4.6c, and

4.7c). The third type is a single crest between the tips of the metaconid and protoconid

DHs, with or without accompanying minor crests (Figs. 4.6e, 4.7b). In the fourth type,

mesial and middle crests (either both complete or one interrupted) link the protoconid and

metaconid DHs (Figs. 4.6f,g, 4.7d). Other minor variations in the study sample are not

discussed here (e.g., Fig. 4.6d,h).

Homo neanderthalensis exhibits the most prominent trigonid crest expression at

the EDJ, characterized in some specimens by a single sharp crest between the metaconid

and protoconid DHs with no other associated crest features. Pan t. verus is most variable

in TCP, while P. robustus, A. africanus, and G. blacki present little in the way of trigonid

crest morphology. The relationship between the informal types of TCP above and two

other discrete dental traits, mesial and distal trigonid crests (Korenhof, 1982, Sasaki and

Kanazawa, 1999), is addressed below.

In unworn teeth, the TCP can, in most cases, be inferred accurately from the OES;

however, moderate wear can make the interpretation of TCP difficult. In worn molars, the

EDJ preserves trigonid crest features that form during the development of the tooth.

Generally, our analysis supports the conclusion that trigonid crest pattern is primary-

definitive in nature, with crest features present at the OES associated with matching crest

features at the EDJ. Even small accessory crests observable at the OES have dentine

analogues.

119

Protostylid

Variation in protostylid expression at the EDJ is summarized schematically in

Figure 4.8. In this schematic representation, we have included morphological features on

the mesial border of the protoconid DH and on the distal border of the hypoconulid DH.

In minor forms of protostylid expression (Figs. 4.8b,c, 4.9a), small wrinkles/depressions

are present at the EDJ. Such features may be located mesially on the slope of the

protoconid DH, centrally between the protoconid and hypoconid DHs, and/or distally

between the hypoconid and hypoconulid DHs. When the protostylid is strongly

expressed, the cingular shelf is large, and it is the shape of the buccal slopes of the

protoconid and hypoconid DHs that dictate the morphology of the buccal crown surface

(Figs. 4.8d–f, 4.9b–d). We see no reason to exclude mesial and distal cingular features

from the “protostylid” complex, and thus our analysis differs from some previous studies

of this trait (e.g., Hlusko, 2004). Examination of the EDJ of molars in this sample

suggests that these morphological features are the result of the same developmental

processes. Taxa with marked protostylid expression include A. africanus, P. robustus,

and G. g. beringei. All of the other taxa present minor expression of the protostylid (with

the exception of the single G. blacki molar, which presents no protostylid morphology).

All of the OES structures included as a protostylid originate at the EDJ, with only

a minor influence due to the differential deposition of enamel. In almost all cases, there is

a consistent relationship between protostylid manifestations at the EDJ and at the OES,

and therefore the protostylid is a primary-definitive trait. Even when the OES expression

of the protostylid is complex, it is matched by an equivalent morphology at the EDJ (Fig.

4.9d). In a small number of cases, minor surface features on the buccal side of the buccal

120

DHs could not be detected at the OES, but the influence of dental attrition on the

manifestation of this trait at the OES could not be ruled out in these cases.

Discussion

The aim of this study was to address three questions related to the development of

discrete dental traits: Do the four dental traits originate at the EDJ? What is the

contribution of the EDJ to trait expression at the OES? Is the process of trait

development, as inferred from the shape of the EDJ and overlying enamel cap, consistent

among the study taxa? With respect to the first two questions, our results are consistent:

the presence and degree of morphological expression of C6, C7, trigonid crest pattern,

and protostylid are dictated primarily by the EDJ. Enamel deposition rarely masks trait

presence at the EDJ, nor are there any OES traits in the absence of EDJ expression. These

results mean that the EDJ of moderately worn teeth may be used to assess the presence or

absence of traits (e.g., the second C6 in Fig. 4.3b), and in unworn teeth, information

about the EDJ may clarify the developmental basis of traits present at the OES. Our

results demonstrate a strong correlation between the EDJ and OES morphology for the

traits studied here, and they suggest a consistent predictive relationship between EDJ and

OES morphology for the majority of dental traits incorporated into anthropological

analyses (contra Kraus, 1952; Schwartz et al., 1998; Olejniczak et al., 2004).

The traits C6 and C7 are discussed together because they are both accessory

cusps, albeit present in different parts of the tooth crown. In all molars, a feature scored

as a C6 at the OES was located directly above a DH-like feature at the EDJ. In some

cases, this DH was similar in shape to the DHs of the adjacent primary cusps (i.e.,

hypoconulid and entoconid) in its degree of pointedness and slope shape, while in other

121

cases, the DH was more like a tubercle with a low, blunt tip. The resolution of the

microCT scan may influence the morphology of small EDJ features, and this must be

considered in assessments of the original shape of diminutive DHs at the EDJ.

Nonetheless, all C6s in the study sample can be classified as primary-definitive traits

(sensu Nager, 1960), with clear evidence that the trait originates at the EDJ. As with most

cusps, enamel deposition alters the shape of the DH through the creation of more convex

cusp slopes and a blunter, rounded cusp tip.

In all teeth in which the C7 is large in relation to adjacent cusps, there is an

equivalent DH-like feature at the EDJ. Even in teeth in which a small cuspule is located

in the valley between the metaconid and entoconid, there is a corresponding elevation at

the EDJ. Some manifestations of C7 at the OES (those corresponding to a type 1A under

the ASUDAS) are more ambiguous at the EDJ. For example, while a cuspule-like

morphology could be argued for the OES, the corresponding EDJ is marked by a

protuberance rather than a distinct, isolated elevation of tissue that would be expected to

underlie an enamel cuspule. Nonetheless, although concordance between the OES and

EDJ in the metaconulid-type C7 is not as pronounced as the interconulid-type, it is still a

primary-definitive trait under Nager’s classification.

The nature of the developmental processes underlying accessory-cusp formation

relates directly to our third question regarding developmental similarity of traits both

within and between different taxa. Should a hypoconulid-type C6 in a P. t. verus

specimen and a fovea-type C6 in a G. g. beringei specimen be coded as the same discrete

trait for the purpose of phylogenetic analysis? From our observations, these two types

appear to be the result of subtly different developmental processes (as an aside, we

suggest that the term “entoconulid” as a synonym for C6 is inappropriate unless one is

122

specifically highlighting the spatial association of this accessory cusp with the

entoconid). Similarly, the two types of C7, observed between the metaconid and

entoconid, appear to represent different underlying developmental processes. This

conclusion is supported by one A. africanus specimen (STW 560A) that exhibits both a

metaconulid-type and interconulid-type C7 (complicating attempts to classify these two

features under one trait).

Can developmental genetic research throw any light on these results? Recent

research into cusp patterning on murine teeth (Jernvall and Jung, 2000; Jernvall and

Thesleff, 2000) suggests that an accessory cusp at the OES, associated with a DH at the

EDJ, corresponds to a secondary enamel knot, the presence and location of which is

determined by the genetic pathways that control the expression domains of the gene

products (Kassai et al., 2005). It has been suggested that the pattern of primary and

secondary cusps on a mammalian tooth crown is the outcome of an iterative cusp-

patterning program (Polly, 1998; Jernvall and Jung, 2000; Jernvall and Thesleff, 2000;

Salazar-Ciudad et al., 2003), which is influenced both by the genes that control the

spacing and size of DHs and/or the overall size of the crown. Cai et al. (2007) showed

that, in mice and rats, epithelial tissue dictates cusp size and the underlying mesenchymal

tissue dictates crown size. Under this paradigm, it is unlikely that a tooth would exhibit

morphology resulting from a gene for a C6 or double-C6; rather, the pattern of spacing of

secondary enamel knots probably results in the formation of (1) an accessory cusp

adjacent to the hypoconulid and (2) another accessory cusp adjacent to that (Fig. 4.3b).

Similarly, the difference between a metaconulid-type and an interconulid-type C7 may

reflect patterns of growth (or differentiation) rates within the developing tooth germ

rather than a one-to-one relationship between a gene and a feature. Our observations

123

indicate that DH-like features can appear in many locations on the EDJ (e.g., on a

trigonid crest, between the hypoconid and hypoconulid DHs, or on a crest between the

hypoconulid and entoconid DHs). Within a developmental paradigm that incorporates a

patterning-cascade mode of cusp development (sensu Jernvall and Jung, 2000), it may be

difficult to devise a simple coding scheme for accessory cusps that is consistent with their

developmental origin. Examination of EDJ expression of accessory cusps reveals

morphological variation that is less obvious at the OES, and that will need to be

addressed as discrete-trait analysis extends beyond modern humans to other primate taxa.

While the majority of accessory cusps have corresponding DHs underlying them

at the EDJ, in some specimens, there is only a faint corresponding elevation at the EDJ.

In a number of specimens, C6s at the OES (in the range of 0.5–1.0 mm in diameter)

presented morphology that, in terms of cuspule shape, must be attributed to the growth of

the enamel rather than the EDJ template. Histological analyses of hominoid enamel

formation have determined that the ameloblasts deposit more enamel per day over cusp

tips (on average) compared to lateral or cervical regions of the tooth crown (Beynon et

al., 1991). Assuming that DHs of C6s form in a similar manner to the DHs of the primary

cusps (i.e., with the initiation of a secondary enamel knot), then ameloblasts in these

locations may be depositing proportionately thicker enamel than adjacent regions. This

could result in cusplike morphology at the OES and explain the apparent discrepancies

between EDJ shape and the OES expression of accessory cusps seen in some teeth.

Histological analysis of accessory cusps may throw light on the ontogeny of these

features.

Examination of the EDJ proved especially valuable for the assessment and

interpretation of trigonid crest expression in the study sample. Like accessory cusps,

124

trigonid crest patterns present at the OES derive from the EDJ. The high prevalence of a

middle crest on the EDJ of the H. neanderthalensis sample supports conclusions by

Bailey (2002) that the midtrigonid crest is a characteristic feature of this taxon compared

to modern humans. However, more comparative studies are needed before it will be

possible to say whether the manifestation of this trait in H. neanderthalensis is derived or

primitive. Investigation of the EDJ has shown that trigonid crest patterning can be highly

variable, and that sorting this variation into midtrigonid (Wu and Turner, 1993; Bailey,

2002) and a distal trigonid crest traits (Korenhof, 1982; Sasaki and Kanazawa, 1999) may

be unwarranted for some taxa (e.g., P. troglodytes ssp.). Crest features between DHs,

including mesial, middle, and distal trigonid crests, as well as the crest that periodically

forms between the entoconid and hypoconulid DHs (Fig. 4.3d), may be related to

structural properties (e.g., elasticity or turgidity) of the tissues involved in tooth growth

and to their interactions during development. Butler (1956: 60–61) noted that

ridges/crests “are probably produced by tensions set up in the epithelium by the relative

movement of cusps, owing to unequal growth or to changes in the shape of the follicle.”

Crests may reflect the timing and rate of DH growth and their placement with respect to

other DHs on the developing tooth germ.

Our analysis of the EDJ suggests that protostylid expression is influenced by three

primary factors. First is the relative location of the DHs of the main buccal cusps (i.e., the

DHs of the protoconid, hypoconid, and hypoconulid) with respect to the buccal margin of

the tooth crown (e.g., centrally placed versus located near the lingual margin of the crown

base). The relative placement of the buccal DHs can also be influenced by buccal

expansion of the tooth crown base during growth. The second factor is the shape of the

buccal aspect of the protoconid and hypoconid DHs (e.g., the presence of crests running

125

towards the cervical margin of the tooth; Figs. 4.8e,f, 4.9d), and the third factor (though

of a lesser influence) is the thickness and distribution of enamel on the buccal side of the

dentine crown. Thus, protostylid morphology, including crests, depressions, cusps,

cuspules, wrinkles, and fissures, derives from the EDJ with only minor modification

caused by the differential deposition of enamel. A possible exception to the minor

influence of enamel deposition on OES expression of the protostylid is P. robustus, is an

ongoing analysis of a larger sample of molars from this taxon (Skinner et al., unpub.

data), which indicates that the relatively thick enamel of this taxon can mask EDJ

expression in some cases. In answer to our third question, protostylid development

appears similar among the study taxa.

If cingular remnants and furrowlike features on the mesial and distal portions of

the buccal side of the crown are developmentally related, then they should not be

considered as separate traits for the purpose of dental trait analysis (contra Hlusko, 2004).

Robinson (1956: 199–120) considered the protostylid a complex character in South

African fossil hominins due to “remnants of a cingulum farther back on the buccal

surface as well as on the whole of the mesial face of the crown.” The complexity of this

trait within the hominoid clade has also been noted by Bailey and Wood (2007). If further

evidence supports developmental nonindependence among mesial, central, and distal

components of protostylid morphology, this trait could prove to be difficult to objectively

and meaningfully subdivide into an ordinal scale. Examination of crown size and DH

size/spacing, which are thought to be under separate genetic control (Harris and Dinh,

2006; Cai et al., 2007), may lead to a better understanding of protostylid variation within

and among taxa.

126

Further research will explore the interaction between DH height and placement,

the overall size of the molar crown, and the collective influence of all of these factors on

the expression of C6s, C7s, trigonid crest pattern, and protostylids. We agree with Bailey

and Wood (2007) that new scoring criteria need to be developed for several of the traits

relevant to the study of early hominins, and we suggest that simultaneous examination of

the EDJ in addition to the OES may help in the identification of independent discrete

traits with a demonstrably similar pattern of development.

Conclusion

The results of this analysis indicate that the presence and degree of morphological

expression of four dental traits (C6, C7, trigonid crest pattern, and protostylid) is largely

determined by the factors controlling the shape of the EDJ, and that differential enamel

deposition does not significantly alter the morphology of these traits or create traits at the

outer enamel surface that are not present at the EDJ. For some traits, such as the

protostylid and trigonid crest pattern, EDJ expression reveals considerable variation

within nonhuman primate taxa that will require new trait descriptions and standardized

classifications for comparison within and between taxa. In some cases, EDJ morphology

suggests that traits that appear similar at the enamel surface in different taxa may not be

the result of similar patterns of development, thus limiting their utility for phylogenetic

analysis. Furthermore, because of the significant morphological contribution of the EDJ

to trait expression at the OES, the EDJ of worn teeth may be used as a proxy for the OES

expression of these traits, and this may well result in increases in the size of study

samples of early hominin taxa.

127

Table 4.1. Composition of the lower molar sample. Taxon n M1 M2 M3 Source

P. t. verus 19 6 13 MPI-EVA

G. g. beringei 2 1 1 NMNH

P. pygmaeus ssp. 3 3 ZMB

G. blacki 1 1 SFN

H. sapiens 7 3 4 NMNH, MPI-EVA

H. neanderthalensis 4 4 MNCN

P. robustus 4 3 1 TM, UW

A. africanus 4 1 2 1 TM, UW

Source codes: MPI-EVA, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany; NMNH, National Museum of Natural History, Washington, DC, USA; ZMB, Museum für Naturkunde, Humboldt Universität, Berlin, Germany; SFN, Senckenberg Forschungsinstitut und Naturmuseum, Frankfurt, Germany; MNCN, Museo Nacional de Ciencias Naturales, Madrid, Spain, TM, Transvaal Museum, Pretoria, South Africa; UW, University of Witwatersrand, Johannesburg, South Africa.

128

Table 4.2. C6 frequency (as a ratio) by taxon and tooth type1.

C6 Absent Fovea Hypoconulid Double-F

2 Double-H

2

P. t. verus 4/19 12/19 3/19 G. g. beringei 1/2 1/2 P. pygmaeus ssp. 2/3 1/3 G. blacki 1/1 H. sapiens

3 4/7 1/7 1/7

H. neanderthalensis 3/4 1/4 P. robustus 1/4 1/4 2/4 A. africanus 3/4 1/4 1 Pooled analysis of all molar types (M1–3). 2 Double-F: double fovea-type; Double-H: double hypoconulid-type. 3 One H. sapiens molar presented both a fovea-type and a hypoconulid-type C6

129

Figure 4.1. Virtual reconstruction of a Gorilla gorilla beringei lower left second molar highlighting the four discrete traits examined in this study: cusp 6, cusp 7, protostylid, and trigonid crest pattern (identified by black circles). The cusp 6 in this molar could be considered a double cusp 6 (discussed in text).

130

Figure 4.2. Schematic representation of C6 morphology present at the EDJ in the study sample. View is towards the distal face of the EDJ surface between the DHs of the hypoconulid (Hypd) and entoconid (Ent). (a) No C6 manifestation at the EDJ; (b) single hypoconulid-type C6 manifested as a DH-like feature on the lingual ridge of the hypoconulid; (c) double hypoconulid-type C6 manifested as two DH-like features on the lingual ridge of the hypoconulid DH; (d) single fovea-type C6 manifested as a DH-like feature between the hypoconulid DH and entoconid DH with no tendency for spatial association to either; (e) double fovea-type C6 manifested as two DH-like features between the hypoconulid DH and entoconid DH; (f) C6 complex exhibiting a single hypoconulid-type and single fovea-type DH between the hypoconulid DH and the entoconid DH.

131

Figure 4.3. Selected examples of C6 expression at the EDJ in the study sample (OES of each specimen is inset in top left corner). Abbreviations are: Ent = entoconid, Hyp = hypoconid, Hypd = hypoconulid. (a) Single hypoconulid-type C6 on a lower second molar of P. t. verus (TAI 15012); (b) double hypoconulid-type C6 on a lower second molar of P. t. verus (TAI 11800); (c) single fovea-type C6 on a lower first molar of A.

africanus (STW 421B); (d) DH-like feature located on a crest joining the entoconid and hypoconulid DHs of a lower second molar of G. g. beringei (NMNH 543034). The status of this feature as a manifestation of C6 is unclear. Images are not to scale.

132

Figure 4.4. Schematic representation of C7 morphology present at the EDJ in the study sample. View is towards the lingual face of the EDJ surface between the DHs of the metaconid (Me) and entoconid (Ent). (a) No C7 manifestation at the EDJ; (b) moderately pronounced shoulder on the distal ridge of the metaconid DH; (c) metaconulid-type C7 on the distal shoulder of the metaconid DH, which in some cases can resemble a small DH-like feature; (d) a second example of a metaconulid-type C7 in which a DH-like feature is not closely associated with the metaconid DH; (e) interconulid-type C7 with a DH-like feature on the distal ridge (but separated from the shoulder by a trough) of the metaconid DH; (f) interconulid-type C7 with a DH-like feature at the low point on the ridge between the metaconid DH and entoconid DH.

133

Figure 4.5. Selected examples of C7 expression at the EDJ in the study sample (OES of each specimen is inset in top left corner). Abbreviations are: Me = metaconid and Ent = entoconid. (a) Metaconulid-type C7 on the distal shoulder of the metaconid DH of a lower third molar of P. t. verus (TAI 11790); (b) metaconulid-type C7 farther removed from the metaconid DH of a lower second molar of G. g. beringei (NMNH 543034); (c) interconulid-type C7 on a lower second molar of H. neanderthalensis (SD 756); (d) interconulid-type C7 on a lower second molar of G. blacki (CA 736). Images are not to scale.

134

Figure 4.6. Schematic representation of TCP present at the EDJ in the study sample. View is towards the occlusal surface of the EDJ including the mesial marginal ridge (mmr) and the tips of the DHs (represented as solid black circles) of the protoconid (Pr) and metaconid (Me). (a) Small, minor crests associated with the Pr and Me DHs; (b) multiple large crests running towards the occlusal basin; (c) pronounced but separate crests that extend across the occlusal basin; (d) single pronounced crests running medially towards each other but not joined in the occlusal basin; (e) a single pronounced crest between the Pr and Me DHs with additional less pronounced crests also present; (f) a mesial crest and a middle crest, one of which is not complete; (g) a mesial (originating at or mesial to the DHs) and middle crest that are complete; (h) a middle crest with a small secondary crest in the occlusal basin. This schematic does not exhaust the manifestations of trigonid crest pattern seen in the study sample.

135

Figure 4.7. Selected examples of TCP expression at the EDJ in the study sample (OES of each specimen is inset in top left corner). Abbreviations: Pr = protoconid, Me = metaconid, mmr = mesial marginal ridge. Crest features are adjacent to white stars. (a) A large crest from the protoconid DH with multiple small crests on the metaconid DH (lower second molar of A. africanus, STW 424); (b) single, pronounced middle crest (lower first molar of H. neanderthalensis, SD 540); (c) two crests from the metaconid DH and one crest from the protoconid DH that do not join (lower second molar of P. t. verus, TAI 11779); (d) mesial and middle crests (lower first molar of P. t. verus, TAI 13433). Images are not to scale.

136

Figure 4.8. Schematic representation of protostylid variation present at the EDJ in the study sample. View is towards the buccal face of the EDJ including the buccal margin, the tips of the DHs (represented as solid black circles) of the protoconid (Pr), hypoconid (Hyp), and hypoconulid (Hypd), and the marginal ridge that runs between them. (a) No protostylid morphology along the buccal margin of the EDJ crown surface; (b) minor furrow on the mesial surface of the protoconid DH; (c) similar to (b) and a second furrow on the distal surface of the hypoconulid DH; (d) similar to (c) with a cingulumlike furrow between the protoconid DH and hypoconid DH; (e) pronounced furrow running from the mesial border of the protoconid DH to the mesial surface of the hypoconid DH with a furrow on the distal surface of the hypoconulid DH (small crests are variably manifest on the surface of the protoconid DH); (f) a continuous, cingulumlike furrow across the whole buccal margin of the EDJ in association with crests on the surfaces of the buccal DHs. This schematic is not exhaustive of the manifestations of protostylid morphology present in the study sample.

137

Figure 4.9. Selected examples of protostylid expression at the EDJ in the study sample (OES of each specimen is inset in top left corner). Abbreviations are: Pr = protoconid, Hyp = hypoconid, Hypd = hypoconulid. Features associated with the protostylid are highlighted by white solid lines. (a) Minor furrows extending across the buccal face of the protoconid DH and below the hypoconulid DH (lower second molar of P. t. verus (TAI 13437); (b) cingulumlike furrows extending across most of the buccal face (lower second molar of G. g. beringei, NMNH 543037); (c) prominent furrows below the hypoconulid DH and between the hypoconid and protoconid DHs (lower second molar of P. robustus, SK 3974); (d) prominent furrows along the mesiobuccal face of the EDJ with small tubercle features below the hypoconid DH that are also expressed on the enamel surface (lower second molar of A. africanus, STW 560E). Images are not to scale.

138

CHAPTER 5: PROTOSTYLID EXPRESSION AT THE OUTER ENAMEL

SURFACE AND ENAMEL-DENTINE JUNCTION OF LOWER MOLARS OF

PARANTHROPUS ROBUSTUS AND AUSTRALOPITHECUS AFRICANUS Abstract

Distinctive expressions and incidences of discrete dental traits at the outer enamel

surface (OES) form part of the diagnosis of many early hominin taxa. It has been argued

that one of these discrete dental traits, the protostylid, can be used to discriminate among

southern African Pliocene fossil hominin taxa. However, there is ongoing debate about

A) the extent to which the shape of the enamel-dentine junction (EDJ) determines

protostylid morphology, or whether differential enamel distribution makes a significant

contribution to the morphology of the OES, and B) whether cingulum-like features on the

mesial and distal aspects of the buccal face of mandibular molar tooth crowns are

developmentally independent (and thus should be excluded from comparisons of

protostylid morphology). This study addresses these issues by examining protostylid

expression at the EDJ and at the OES of Paranthropus robustus (N = 22) and

Australopithecus africanus (N = 28) mandibular molars. The results include evidence A)

of statistically significant taxon-specific patterns of protostylid morphology at the EDJ

that are not evident at the OES; B) of non-independence of isolated protostylid features

across the buccal face of the EDJ; C) that differential enamel distribution can reduce

morphological correspondence in protostylid expression between the EDJ and the OES,

and D) that the protostylid retains its taxonomic relevance even in worn teeth.

139

Introduction

Tooth crown morphology has played a central role in the taxonomy and inferred

phylogenetic relationships of Pliocene African hominins. The protostylid, a dental trait

present on mandibular molars, has received particular attention because of its pronounced

expression in a number of teeth attributed to Paranthropus robustus and Australopithecus

africanus (Robinson, 1956; Wood and Abbott, 1983; Suwa et al., 1994; Hlusko, 2004;

Irish and Guatelli-Steinberg, 2003; Guatelli-Steinberg and Irish, 2005). In a recent and

comprehensive analysis of protostylid expression in these taxa Hlusko (2004) concluded

that P. robustus can be distinguished from Au. africanus because the former taxon has a

relatively high frequency of moderate protostylid expression, whereas the latter taxon has

a relatively high number of specimens with either weak or strong expression.

Previous research has demonstrated that EDJ morphology of extant and fossil

hominoid teeth shows both intra- and intertaxonomic differences in dental trait

morphology that are not always evident at the outer enamel surface (Korenhof, 1960,

1961, 1982; Nager, 1960; Kraus and Jordan, 1965; Corruccini and Holt, 1989;

Corruccini, 1998; Schwartz et al., 1998; Sasaki and Kanazawa, 1999; Skinner et al.,

2008). The difficulty of assessing trait morphology is, of course, even more marked when

wear has removed details of the morphology at the OES. However, the extent to which

the shape of the EDJ determines protostylid expression, and the role played by

differential enamel distribution on the expression of the protostylid at the outer enamel

surface (OES) remain unclear. It is also unclear whether cingulum-like features across the

mesial, central, and distal aspects of the buccal face of mandibular molar tooth crowns

are developmentally independent, and should be incorporated into definitions and scoring

of protostylid expression (Hlusko, 2004; Skinner et al., 2008).

140

In this study we assess protostylid expression in P. robustus and Au. africanus at

the EDJ and the OES to address the following questions. Is there an EDJ equivalent of an

OES protostylid? Are current definitions and scoring systems of the protostylid trait

consistent with morphological features expressed at the EDJ? Are there species-specific

differences in any EDJ protostylid morphology between P. robustus and Au. africanus

that are not evident at the OES? Finally, in worn teeth can protostylid morphology at the

EDJ be used for the purposes of taxonomy and phylogenetic reconstruction in place of its

OES expression?

Materials and Methods

Study sample

The study uses fossils from collections housed at the Flagship Museum (formerly

the Transvaal Museum) in Pretoria and at The University of Witwatersrand in

Johannesburg, South Africa. First, second, and third mandibular molars (M1, M2 and M3)

were examined (Table 5.1). Molars attributed to P. robustus come from the sites of

Swartktrans, Gondolin and Drimolen (Robinson, 1956; Tobias et al., 1977; Grine 1989;

Menter et al., 1999; Keyser et al., 2000; De Ruiter, pers. comm. 2006) and those

attributed to Au. africanus come from the site of Sterkfontein (Moggi-Cecchi et al.,

2006). Taxonomic affiliations and information about tooth type were taken from these

publications.

The specimens included in this study represent a subset of mandibular molars of

P. robustus and Au. africanus. This is for two reasons. First, only isolated teeth (or teeth

from small mandibular fragments) could be scanned with the portable micro-computed

tomography scanner used for this study. Second, in some of the isolated teeth differential

141

mineralization during fossilization has resulted in the enamel and dentine having similar

levels of radiopacity. In these cases the inability to segment the two tissues prevents

visualization of the EDJ.

EDJ and OES surface reconstruction

Each tooth was imaged by a micro-computed tomography scanner (SKYSCAN

1172, Kontich, Belgium) at an isometric voxel resolution of 14 µm (100Kv, 94mA,

2.0mm aluminum and copper filter, 0.12 rotation step, 360 degrees of rotation, 2 frame

averaging). Raw projections were converted into TIFF image stacks using NRecon

(parameters: ring artifact correction = 10; beam hardening = 30%). To reduce the size of

the resulting files, teeth were downsampled to 30 microns using Amira (triangle filter,

v4.1, www.amiravis.com). To facilitate tissue segmentation, the complete image stack for

each tooth was filtered using a three-dimensional median filter (kernel size of 3) followed

by a mean of least variance filter (kernel size of 3), implemented as a computer-

programmed macro. This filtering process results in more homogenous tissue classes

(e.g., enamel vs. dentine) and allocates pixels with intermediate gray-scale values at

tissue interfaces (i.e., air-enamel, enamel-dentine, air-dentine) to the appropriate tissue

(Schulze and Pearce, 1994).

Filtered image stacks were imported into the Amira software package and enamel

and dentine tissues were segmented using the 3D voxel value histogram and its

distribution of gray-scale values, which typically presents a trimodal distribution with one

peak representing dentine, another peak representing enamel, and a third peak

representing air and background noise in the images. In fossil teeth where the enamel and

dentine differ substantially in their degree of mineralization (and therefore their densities,

142

and thus also in the ability of X-rays to pass through them), the filtering process results in

gray-scale pixel value distributions for each tissue that do not overlap. In other teeth,

diagenetic alteration (e.g., dentine remineralization) may result in similar tissue densities

and thus overlapping gray-scale pixel value ranges for enamel and dentine (Olejniczak

and Grine, 2006). Only teeth in which there was a clear separation of enamel and dentine,

resulting in well-distinguished gray-scale values and accurate representations of the EDJ,

were used in the study. After segmentation, the EDJ is reconstructed as triangle-based

surface model using Amira (surface generation module using unconstrained smoothing

parameter). In specimens that preserved only the enamel cap, a surface model of the EDJ

was created by digitally removing the occlusal surface of the reconstructed enamel cap

surface model.

Defining the protostylid based on EDJ morphology

A protostylid was described by Dahlberg (1950:16) as “an elevation or ridge of

enamel on the anterior part of the buccal surface of the lower molars, which ascends from

the gingival end of the buccal groove and extends mesio-occlusally.” The Arizona State

University Dental Anthropology System (ASUDAS) defines the protostylid as a

paramolar cusp found on the buccal surface of the protoconid that is normally associated

with the buccal groove (Turner et al., 1991). Expression states under the ASUDAS

system focus on a secondary groove that extends mesially from the buccal groove and

culminating, in marked expression, as a cusp with a free apex. In the most recent and

comprehensive analysis of protostylid expression in fossil hominins (Hlusko, 2004) a

variable groove, or protoconid shelf, on the mesial aspect of the protoconid (e.g., visible

in Type 1 in Hlusko’s Fig. 1, 2004: 583) was excluded from the definition of the

143

protostylid. This was because there was not strong evidence indicating morphological

covariation between this feature and morphology of the buccal groove. In order to clarify

what morphological features at the EDJ of southern African fossil hominins should be

included in the definition of the protostylid we examined the expression of all cingulum-

like features on the mesial, central and distal aspects of the buccal face of the EDJ and

OES.

Quantification of protostylid morphology at the OES and EDJ

In order to address any correspondence in protostylid expression between the

OES and EDJ, as well as to test for species differences, the buccal face of each surface

was divided into three regions (Fig. 5.1). The first region (referred to as anterior) begins

at the mesial border of the crown and extends to a plane orthogonal to the long axis of the

tooth and to the occlusal surface that passes through the tips of the two mesial cusps (at

the OES or through the dentine horns). The second region (referred to as middle) extends

from the end of the anterior region to a parallel plane that passes through either the tip of

the OES hypoconid, or its dentine horn. The third region (referred to as posterior) begins

at this latter plane and extends to the distal margin of the crown. Cingulum-like features

(e.g., crests/ridges) on the surface of each specimen within each region were recorded and

measured separately. Measurements were taken using the 3D measurement tool in Amira.

In the case of curved ridges, linear segments were summed along the feature to capture its

length. In order to account for overall size differences both between specimens, as well as

between the OES and EDJ surfaces of each tooth, all measurements were scaled by the

geometric mean (Mosimann, 1970; Jungers et al., 1995) of maximum mesiodistal length,

144

the maximum buccolingual breadth of the mesial portion of the crown, and the maximum

buccolingual breadth of the distal portion of the crown.

Correspondence in protostylid morphology between the EDJ and the OES

The relationship between protostylid expression at the OES and EDJ can be of

two types. On the one hand, protostylid morphology at the EDJ is not substantially

altered by the deposition of enamel. Alternatively, differential enamel deposition can

modify protostylid-like morphology at the EDJ, or create a protostylid at the OES that is

not present at the EDJ. Clearly, the formal definition of the protostylid and the ability to

score the feature at the OES would be affected if the relative contribution of the EDJ and

enamel cap to trait expression at the OES differs within, or between, taxa.

Correspondence within regions and across the whole buccal face was assessed

quantitatively by deriving the ratio of crest(s) length at the EDJ divided by crest(s) length

at the OES (multiplied by 100).

Species-specific differences in protostylid morphology

Previous analyses of protostylid expression at the OES between P. robustus and

Au. africanus noted differences in the degree of expression, rather than in the kind of

protostylid (Robinson, 1956; Hlusko, 2004). Skinner and colleagues (2008) noted

evidence for species-specific differences in the EDJ morphology of other dental traits

(e.g., for cusp 6 and trigonid crest pattern) that were difficult to detect at the OES. In this

examination of protostylid-like EDJ morphology in P. robustus and Au. africanus, we

assess whether there are differences in the degree of trait expression, and/or whether the

145

taxa display different patterns of protostylid expression at the EDJ that would suggest

differences in the development of the trait.

Taxonomic comparison included both visual inspection of OES and EDJ

expression of protostylid morphology, as well as, a statistical comparison of the overall

length (and length within each of the three regions of the buccal face defined above) of

protostylid features at each surface. After conducting a Kruskal-Wallis ANOVA to

determine there were no significant differences between molar types for each variable,

molars were grouped for the purpose of comparing species differences. Due to unequal

variances between species for a number of measured variables a non-parametric Mann-

Whitney U test was used to compare group medians. Differences are considered

statistically significant at p-values at or below 0.05. In the case of antimeres the better

preserved side was used.

Results

Defining the protostylid based on EDJ morphology

Morphology at the EDJ does not fit well into current definitions of the protostylid

that were devised by studying its expression at the OES. There are two major areas of

mismatch. First, many teeth present single or multiple crest-like features in the anterior,

central, and posterior regions of the buccal face of the EDJ that appear to be linked

morphologically. In Figures 5.2 and 5.3 we present a range of cingular features on the

buccal face of the EDJ of P. robustus and Au. africanus, respectively. In the anterior

region on molars from both taxa, crests are often present that are continuous with crests

in the middle region (e.g., Fig. 5.2e,f, 5.3c,d,e). Crests in the posterior region that run

from the tip of the hypoconulid dentine horn inferomedially are common in both taxa

146

(Figs. 5.2a-f, 5.3a-f). In many cases, such crests appear to represent the distal portion of a

more, or less, continuous cingulum (e.g., Fig. 5.2a. 5.3e) suggesting that these features

are developmentally linked. These findings contradict current definitions of the

protostylid in which only centrally-located features can be incorporated into OES-based

definitions of the protostylid.

The second discrepancy between the OES-based definitions and what is observed

at the EDJ is that the buccal groove seen at the OES is not associated with any distinct

structure at the EDJ, yet the form of the buccal groove at the OES (e.g., a mesially

running secondary groove, or a V-shaped groove) is central to most classificatory

schemes of protostylid expression. The buccal groove at the OES marks where the

mineralizing fronts derived from the tips of the protoconid and hypoconid dentine horns

meet, and its form is likely influenced by the depth and morphology of the concave EDJ

surface between the two, whereas the protostylid morphology on the EDJ in this region

tends to be a cingulum-like crest between the two dentine horns (Fig. 5.2b, 5.3d).

Correspondence in protostylid morphology between the EDJ and OES

Across the whole buccal face correspondence between the EDJ and OES in Au.

africanus tends to be greater than in P. robustus although this taxonomic difference is not

statistically significant (Table 5.2). When the anterior and middle regions are combined

(crests tend to be continuous between these regions) there is significantly less

correspondence between the OES and the EDJ in P. robustus than in Au. africanus (p =

0.02). This difference is because marked crest-like features between the protoconid and

hypoconid dentine horns present at the EDJ of P. robustus are rarely matched in size or

shape at the OES, and because of the strong correspondence between the OES and EDJ in

147

mesially-located crest features in Au. africanus. In the posterior region both taxa exhibit a

similar lack of correspondence with crest-like features at the EDJ being much less

prominent at the OES.

Species-specific differences in protostylid morphology

While the EDJ expression of protostylid morphology between P. robustus and Au.

africanus does not differ significantly when taken along the whole length of the buccal

face of the EDJ, the regional distribution of these crest features does differ significantly

(Table 5.2, Fig. 5.4) between the taxa. For example, P. robustus exhibits significantly

greater (p = 0.04) protostylid expression in the middle region (i.e., between the

protoconid and hypoconid dentine horns), while Au. africanus exhibits significantly

greater (p = <0.001) expression in the anterior region (i.e., mesial to the protoconid

dentine horn). Both taxa exhibit crest-like features in the posterior region.

These differences form a qualitative difference in the pattern expression between

the taxa that is apparent at the EDJ. That is Au. africanus molars (an exception is

STW412A) tend to exhibit a crest-like feature at the EDJ that extends to the mesial side

of the protoconid dentine horn, whereas P. robustus molars (an exception is SK104)

exhibit crest-like features that extend no further than immediately below, or slightly distal

to the protoconid dentine horn. The specimens from Drimolen and Swartkrans are

consistent in their expression of the P. robustus pattern. There is also a difference in

where the cingular ridge terminates in the central region of the buccal face of the EDJ. In

Au. africanus it terminates between the protoconid and hypoconid dentine horns with the

crest often dropping inferiorly towards the cervix (Fig. 5.3d,e, 5.5a,b). This contrasts with

the most common pattern in P. robustus, which is for the crest to extend distally and

148

superiorly towards the tip of the hypoconid dentine horn to form a crescent pattern (Fig.

5.2b,c,e).

Protostylid expression at the OES reveals a slightly different pattern of species

differences than at the EDJ. Similar to the pattern noted above for the EDJ, the OES of

Au. africanus exhibits significantly longer crest features in the anterior region (p =

<0.001). These anterior crest features, along with the lack of crest expression at the OES

in the central region, contribute to a significant difference in crest length along the whole

buccal face of the OES (p = 0.04).

Protostylid expression in worn fossil teeth

In Figure 5.5 we illustrate the OES and EDJ of four partially worn molars. While

it is very difficult to detect any protostylid morphology at the OES in three of the molars

(Fig. 5.5a,c,d), there is clear evidence of a protostylid equivalent at the EDJ. Furthermore,

the species differences outlined above are evident at the EDJ, with the Au. africanus

specimens showing a mesially-extended crest (Fig. 5.5a,b,d), whereas the P. robustus

specimens have a marked crest between the protoconid and hypoconid dentine horns

(Fig. 5.5c). It is notable that in STW520 (Fig. 5.5b), which has EDJ evidence of a

protostylid, the Au. africanus pattern is not detectable at the OES even though wear does

not appear to have significantly altered this region of the crown surface.

Discussion

We set out to relate current definitions and scoring systems of the protostylid

dental trait, which have been based on the study of the OES, with what can be observed

at the EDJ in the same location. In these Pliocene hominin taxa the protostylid at the EDJ

149

is in the form of a crest, or crests, along the buccal face of the crown below the dentine

horns of the buccal cusps. As noted in a previous publication (Skinner et al., 2008) the

presence, size and shape of the protostylid in hominoids is influenced by the size, shape

and spacing of the dentine horns, the overall size of the molar crown, and the distribution

of enamel. The taxonomic differences between the frequent expression at the EDJ in Au.

africanus of a mesially-extended crest, versus marked crest expression between the

protoconid and hypoconid dentine horns in P. robustus, are likely the result of differences

in the interaction of the developmental processes that control the spacing of the buccal

dentine horns and the relative width of the crown (Polly, 1998; Jernvall and Jung, 2000;

Jernvall and Thesleff, 2000; Kassai et al., 2005). Note for example in Figure 5.3d and

5.3e, the relatively central position of the buccal dentine horns and the pronounced crest

features along the buccal margin of the EDJ. The morphology that appears to characterize

the P. robustus and Au. africanus patterns should not, in our opinion, be considered as

different manifestations of a single feature along a morphological continuum of minor to

marked expression. Although they are both manifestations of a cingulum feature

resembling a crest, the crest-like feature is in different parts of the crown. It is also

apparent that enamel thickness, which from a whole crown perspective is greater in P.

robustus (Olejniczak et al., nd), appears to have more influence on the OES manifestation

of protostylid morphology in P. robustus than it does in Au. africanus.

The protostylid can be present on the buccal face, in the distal, central, and mesial

regions and its definition should not be limited to the region of the buccal groove

(Robinson, 1956; but contra Dahlberg, 1950; Hlusko, 2004). Crest features along the

whole buccal face of the EDJ appear to be the result of the same developmental

processes. Furthermore we suggest that it may be incorrect to include a cusp feature in

150

the definition of the protostylid; particularly for the taxa included in this study. In none of

the teeth in our sample did we find a dentine horn-like feature underlying a protostylid

feature at the OES. While true cusps may form in the region adjacent to the protoconid,

we suggest that these are the result of the formation of a secondary enamel knot, and not

an especially pronounced expression of the protostylid, which as we have suggested

above is a cingulum-derived crest feature. Thus, a cusp should not be considered as the

end point on the continuum of protostylid expression. The association of the protostylid

trait primarily with the buccal groove at the OES and the division of expression into “v-

shaped” furrows, a “secondary groove,” or a “paramolar cusp” are not warranted for

these taxa (nor for many other hominoid taxa; see images in Skinner et al., 2008). The

fidelity, homology, and consistency of expression of these variants need to be

investigated more thoroughly before they can be included in definitions of this trait.

Any scoring system of protostylid expression equivalent to the ASUDAS for

variations within and among modern human populations for the early hominin taxa we

have investigated has to recognize the considerable variability in the presence and

morphology of crest features along the buccal face of the dentine crown. Converting this

variability into a graded continuum of expression, for the purpose of comparison among a

range of taxa, may prove difficult. Scoring systems may need to be devised for different

taxonomic comparisons and we encourage further examination of EDJ expression of

protostylid morphology and other dental traits in order to establish appropriate definitions

and scoring criteria for hominin taxa (Hlusko, 2004; Bailey and Wood, 2007; Martinón-

Torres et al., 2007).

We found that the OES morphology does not faithfully replicate the EDJ

morphology, and this suggests that differential enamel deposition contributes to the

151

differences in how the protostylid is expressed at the OES in Au. africanus and P.

robustus. In unworn teeth with strongly expressed protostylid morphology, the OES may

give a reasonable representation of what is present at the EDJ, however, as the degree of

expression decreases and/or enamel thickness increases the reliability of the OES for

accurately comparing protostylid morphology between these taxa is reduced. Visualizing

the EDJ manifestation of this trait presents a new opportunity to improve our

understanding of its development, our methods for comparing its presence, and the

inferences that are drawn based on its variable manifestation in the fossil record. Trait

definitions and scoring systems may require reference to both the OES and EDJ to insure

their homology and maximize their potential for taxonomic and phylogenetic

applications.

The concerns we have expressed regarding the definition and scoring of the OES

protostylid feature do not negate the many previous studies that have noted differences in

trait presence between the study taxa. Robinson (1956) noted a tendency for a better-

developed protoconidal cingulum in Au. africanus than in P. robustus. Wood and Abbott

(1983) noted a greater frequency of protostylid expression in P. robustus, but suggested

that when a protostylid was present in Au. africanus, its expression was more marked.

Hlusko (2004) noted that with respect to the protostylid at the OES, Au. africanus tends

toward a bimodal distribution, with either no expression or a high degree of expression,

while P. robustus has a more normally distributed expression of the protostylid.

Consequently, Hlusko (2004) argues that it is frequency distribution that separates Au.

africanus and P. robustus and not the central tendency, but Hlusko’s definition of the

protostylid excluded isolated more mesial crests which may influence this apparent

taxonomic distinction. For example, STW 309A and STW 246 that are given as examples

152

of no protostylid expression (Type 1) and minor protostylid expression (Type 2)

respectively (Hlusko, 2004:583), both possess marked mesial crests that we would argue,

based on the results of this study, should be included in any assessment of protostylid

expression. Guatelli-Steinberg and Irish (2005) also found that protostylids in Au.

africanus mandibular first molars were more strongly expressed than in P. robustus. Our

results are consistent with these previous studies in suggesting taxonomic differences in

both degree and kind of protostylid expression with significant differences present in both

the length and location of crests. A forthcoming study will address the relationship

between protostylid expression at the OES and the EDJ in P. boisei from East Africa, and

what that implies for ongoing investigations into the phylogenetic relationship among Au.

africanus, P. robustus, and P. boisei.

Conclusion

Analysis of images of the EDJ obtained from teeth belonging to Au. africanus and

P. robustus suggests that the protostylid features at the OES correspond to crest-like

features along the buccal margin of the EDJ. We also suggest that these crest-like

features, be they mesial, centrally-located, and/or distal, appear to be developmentally

linked, and that researchers should be cautious about selectively excluding any of these

features some for the purpose of scoring protostylid expression, as the location of crests

may differentiate taxa. Differential enamel deposition can alter the EDJ morphology,

particularly in the thicker-enameled molars of P. robustus. This means that protostylid

expression at the OES cannot always be used as a proxy for the equivalent EDJ

morphology and that the pattern that characterizes a taxonomically relevant expression of

the trait at the OES might include contributions from both dentine and enamel.

153

A significant difference in the EDJ morphology of the protostylid is present

between Au. africanus and P. robustus. In Au. africanus the crest-like structures at the

EDJ extend mesially of the protoconid dentine horn, whereas in P. robustus the crest-like

structures are more pronounced between the dentine horns of the protoconid and

hypoconid. These differences enable teeth from these two taxa to be differentiated even

when wear has removed the OES evidence of the protostylid.

154

Table 5.1. Au. africanus and P. robustus mandibular molars included in this study. Au. africanus Tooth Site P. robustus Tooth Site

STS 9 LRM1 Sterkfontein DNH 60B LRM1 Drimolen STW 145 LRM1 Sterkfontein DNH 67 LRM1 Drimolen STW 291 LRM1 Sterkfontein SK 104 LRM1 Swartkrans STW 309A LRM1 Sterkfontein SK 828 LLM1 Swartkrans STW 364 LRM1 Sterkfontein SK 843 LLM1 Swartkrans STW 421A LRM1 Sterkfontein SK 846A LRM1 Swartkrans STW 421B LLM1 Sterkfontein SK 3974 LRM1 Swartkrans STW 3 LLM2 Sterkfontein DNH 60C LRM2 Drimolen STW 213 LLM2 Sterkfontein SK 1 LLM2 Swartkrans STW 213 LRM2 Sterkfontein SK 6 LRM2 Swartkrans STW 308 LRM2 Sterkfontein SK 843 LLM2 Swartkrans STW 412A LRM2 Sterkfontein SK 1587B LRM2 Swartkrans STW 412B LLM2 Sterkfontein DNH 75 LRM3 Drimolen STW 424 LLM2 Sterkfontein SK 6 LRM3 Swartkrans STW 537 LLM2 Sterkfontein SK 22 LRM3 Swartkrans STW 537 LRM2 Sterkfontein SK 75 LRM3 Swartkrans STW 560D LLM2 Sterkfontein SK 841B LLM3 Swartkrans STW 560E LRM2 Sterkfontein SK 851 LRM3 Swartkrans STS 55B LLM3 Sterkfontein SKX 5002 LLM3 Swartkrans STW 280 LRM3 Sterkfontein SKX 5014 LRM3 Swartkrans STW 491 LLM3 Sterkfontein SKX 10642 LLM3 Swartkrans STW 520 LRM3 Sterkfontein SKX 10643 LRM3 Swartkrans STW 529 LLM3 Sterkfontein STW 537 LRM3 Sterkfontein STW 537 LLM3 Sterkfontein STW 560A LRM3 Sterkfontein STW 560B LLM3 Sterkfontein TM 1520 LLM3 Sterkfontein

155

Table 5.2. Comparison of correspondence1 between the EDJ and OES and mean

2 protostylid crest length at the EDJ and OES.

Variable P. robustus n Au. africanus n Mann-Whitney U3

Correspond. – anterior + middle 230.8 ± 169.3 15 117.3 ± 31.1 13 p = 0.02 Correspond. – posterior 203.1 ± 81.3 7 239.5 ± 142.4 9 Ns Correspondence – whole buccal face 268.6 ± 207.1 15 172.9 ± 110.6 14 Ns EDJ – anterior 2.4 ± 6.0 19 18.8 ± 9.4 20 p = <0.001 EDJ – middle 32.0 ± 10.5 19 23.1 ± 15.0 20 p = 0.04 EDJ – posterior 18.4 ± 11.7 19 19.3 ± 7.3 20 Ns EDJ – whole buccal face 52.8 ± 19.6 19 61.3 ± 22.0 20 Ns OES – anterior 1.6 ± 5.6 15 16.1 ± 12.1 16 p = <0.001 OES – middle 18.7 ± 7.7 15 18.2 ± 14.7 16 Ns OES – posterior 5.5 ± 6.6 15 6.6 ± 8.3 16 Ns OES – whole buccal face 25.9 ± 13.7 15 40.9 ± 23.6 16 p = 0.04 1. Each species value represents the mean of the ratio of crest length between the EDJ and OES for each specimen for a given portion of the tooth crown. 2. Each species value represents the mean of crest length scaled by a geometric mean of crown size for the EDJ and OES respectively. 3. While means for each variable are reported the Mann Whitney U test specifically tests whether the two groups were likely to come from populations with the same median values (ns = not statistically significant at or below p = 0.05)

156

Figure 5.1. Surface model of the EDJ of a mandibular molar illustrating the division of the buccal crown face into three regions: anterior, middle, and posterior (OES surface is inset in top left corner). The dashed line marks which crest features are measured. The OES of each tooth is similarly partitioned and measured.

157

Figure 5.2. EDJ expression of protostylid morphology in six mandibular molars of P.

robustus in both buccal (top) and distal (bottom) view (a buccal view of the OES surface of each tooth is inset at middle left). Note the prominent crests in the middle of the buccal face and the lack of cresting on the mesial side of the protoconid dentine horn. (A) SK1587B_RM2; (B) SK828_LM1 [mirrored]; (C) SK843_LM1 [mirrored]; (D) SK6_RM2; (E) SKX10643_RM3; (F) SK104_LM1 [mirrored].

158

Figure 5.3. EDJ expression of protostylid morphology in six mandibular molars of Au.

africanus in both buccal (top) and distal (bottom) view (a buccal view of the OES surface of each tooth is inset at middle left). Note the prominent crests in the anterior region of the buccal face extending around the base of the protoconid dentine horn. (A) STW309A_LRM1; (B) STW145_LRM1; (C) STW421A_LRM1; (D) STW537_LLM2 [mirrored]; (E) STW560A_LRM3; (F) STW412A_LRM2.

159

Figure 5.4. Relative protostylid crest length at the EDJ and OES in each region of the buccal face of the molar crown. Black bar denotes the mean and shaded bar represents the 95% confidence interval of each variable. Black stars highlight significant differences below p = 0.05.

160

Figure 5.5. OES (top) and EDJ (bottom) expression of protostylid morphology in four mandibular molars exhibiting minor to marked wear. Note that assessing protostylid morphology at the worn enamel surface of these teeth is difficult to impossible. However, the EDJ surface reveals underlying taxon-specific protostylid morphology. (A) STW291_LRM1 [Au. africanus]; (B) STW520_LRM3 [Au. africanus]; (C) SK846A_LRM1 [P. robustus]; (D) TM1520_LLM3 [Au. africanus - mirrored].

161

CHAPTER 6: SUMMARY AND CONCLUSIONS Thesis summary

This thesis focused on an examination of the enamel dentine junction (EDJ) of

lower molars in various extant hominoids and fossil hominins. Collectively, these

manuscripts represent the first comprehensive analyses of EDJ morphology in a range of

hominoid taxa and at the high degree of resolution made possible through the use of

micro-computed tomography. They explore the taxonomic relevance of EDJ morphology,

both quantitatively and qualitatively, and in doing so reveal how detailed aspects of the

outer enamel surface (OES) morphology develop. They also provide insights into the

developmental processes that determine the form of the functional interface between the

dentition and the food, upon which natural selection acts.

The first chapter introduced the aims of the thesis and reviews the relevant

literature. It also provided a detailed discussion of the materials and methods used to

image and analyze EDJ morphology. Chapter two demonstrated that when the

morphology of the EDJ can be captured in sufficient detail, analysis of its morphology

can discriminate between species and subspecies of extant chimpanzees. Chapter three

extended these findings to an analysis of lower molars belonging to Australopithecus

africanus and Paranthropus robustus from southern Africa and demonstrated that EDJ

morphology is distinctive both between each taxon, as well as between first, second, and

third molars of each taxon. Chapter four examined the expression of four dental traits on

the lower molars of a range of extant and extinct hominoids (including fossil hominins).

It demonstrated that these OES traits originate at the EDJ, that the EDJ is primarily

responsible for their degree of expression, and that when examined across a wide range

162

of taxa the morphological variability in the expression of these traits can be considerable.

The fifth chapter focused specifically on one dental trait, the protostylid, and examined

its EDJ manifestation in samples of Australopithecus africanus and Paranthropus

robustus. The results of this analysis revealed taxon-specific patterns in protostylid

expression that are difficult to detect at the OES as well as evidence that current

definitions of the trait itself should be re-examined. The EDJ can be used to address a

number of important questions relating to dental anthropology and paleoanthropology,

and because details of its morphology are preserved even in heavily worn teeth, the EDJ

can play a unique role in generating hypotheses about the taxonomy and systematics of

fossil hominin taxa.

Discussion and Conclusions

Interpretations of EDJ shape differences

The methodology employed in this thesis allows the visualization and analysis of

detailed aspects of both OES and EDJ morphology. In some cases shape differences

between taxa are marked and in some cases they are very subtle. Some may be related to

the function of the tooth (e.g., differences in the relative height of the dentine horns)

while some may reflect random genetic drift (e.g., slight shape differences between Pan

subspecies). Now that high-resolution surface models of the dentine (including the roots

and the dentine component of the crown) and enamel cap can be generated, efforts should

be made to generate and test hypotheses of the functional significance of OES features

(e.g., cingula, accessory cusps, dentine horn shape, crest patterning, dentine crown

height, crown shape, and enamel distribution) using finite element modeling. The

163

consistent shape differences between first, second, and third molars revealed in these

analyses should also be explored with regard to their functional significance. Using large

pedigreed populations, quantitative genetic analysis could reveal the heritability of EDJ

features (e.g., Hlusko and Mahaney, 2003), or based on expectations of population

genetics one could explore whether subtle variation could arise by genetic drift rather

than selection (Weaver et al., 2007).

Selection acting on EDJ and enamel cap morphology

Natural selection can act both on variation of the EDJ, during the growth and

folding of the inner enamel epithelium early in the development of the tooth, and/or on

differences in the way enamel is distributed over the EDJ. This selection will alter the

shape of the features that are interacting with food both when the tooth first erupts into

the mouth and as it wears down. Furthermore, it will alter the way in which the tooth

reacts under different loading conditions and how it resists abrasion. The correspondence

between tooth morphology and diet should be explored, not just with respect to the

enamel surface, but also with regard to EDJ morphology.

Taxonomy and phylogeny

This thesis has confirmed that EDJ morphology can discriminate among major

groups of primates and it has demonstrated the EDJ’s ability to discriminate between

closely related taxa. It may be that the combination of information about the EDJ and the

OES may provide the most effective discrimination between closely related taxa. The

pattern of phylogenetic relationships among the higher primates generated from

164

molecular evidence could also be used to investigate the taxonomic signal of EDJ and

enamel cap morphology. And within the hominin clade the same morphological evidence

could be used to test hypotheses of relationships, such as the proposed monophyletic

relationship among megadont archaic hominins (Paranthropus boisei and Paranthropus

robustus) from East and southern Africa, respectively. Under a model in which

similarities in postcanine crown morphology are the result of convergence, it could be

predicted that EDJ and enamel cap morphology are more similar between P. robustus and

Au. africanus than between the former and P. aethiopicus/P. boisei, while under a model

of shared ancestry the same morphology would be more similar among the megadont

archaic taxa.

What are the implications for dental trait analysis?

Enamel dentine junction morphology may provide information that is independent

of the OES, particularly in those taxa in which the enamel is thick enough to influence

trait expression, and OES trait definitions may need to be refined and scoring systems

amended. There are a number of outstanding issues regarding specific traits that can be

resolved with high resolution images of the OES and EDJ. For example, it could be

determined what lies beneath a so-called “split-hypocone” (Bermúdez de Castro and

Martínez, 1986). The contribution of the EDJ to Carabelli’s trait remains unclear

(Schwartz et al., 1998). Preliminary observations suggest that, like most traits, Carabelli’s

originates at the EDJ, but in minor forms of expression enamel deposition can have a

strong influence on manifestation at the OES. A better understanding of the development

165

of these traits would improve our overall understanding of how teeth grow and our

interpretations of morphological variation.

Understanding EDJ development and testing of developmental models

The growing understanding of the genetics of tooth development has led to the

development of models through which variability in tooth crown morphology can be

interpreted. The applicability of these models, such as those that pertain to cusp

patterning, tooth type determination, or to the relative size of molars (Kavanagh et al.,

2007), to primates should be explored. As most of our understanding of the genetics of

tooth development relates to the early stages of germ development (rather than on the

behavior of enamel-depositing ameloblasts) the EDJ is the ideal structure upon which to

assess the relevance of these models for generating a deeper understanding of the

differences in primate tooth morphology.

The developmental basis of traits also needs to be evaluated. Does an accessory

cusp always need to have an underlying dentine horn? What is the significance of

variation in EDJ/OES crest morphology, and are the variable manifestations discrete, or

are they the consequence of more general properties of the developing tooth germ? The

developmental homology of some traits may not hold up under scrutiny of the EDJ. As

our ability to visualize crown features in detail improves so too does our understanding of

their development as well as their potential relatedness to other crown features.

166

Importance of making images of EDJ morphology widely available

While there have been a number of researchers who have recognized the

importance of EDJ morphology for understanding tooth growth and tooth shape, there are

only two sources, to my knowledge, of numerous images of the EDJ surface of modern

humans (and none in non-human primates). These are Korenhof’s 1960 publication on

upper modern human molars and the book by Kraus and Jordan (1965) on fetal tooth

germs. The answers to many of my own questions regarding tooth crown morphology

were resolved by seeing the images available in these sources. These images, which are

not widely available, are also pertinent to many current debates regarding tooth crown

features. High-resolution computed tomography is becoming increasingly available to

anthropologists and it is a reasonable endeavor, over the next decade, to image the EDJ in

large samples of many extant and fossil primate taxa. Making these images available is

facilitated by the internet and the ability to embed 3D surfaces into digital documents that

can be manipulated by the reader. I believe that great strides could be made in our current

understanding of tooth development, tooth variation and the evolution of dental

phenotypes simply by making these images easily accessible to a wide audience. In many

respects, and for many taxa, the morphology of the EDJ is a tabula rasa. Hopefully, this

will change in the not too distant future.

167

References Avishai, G., Müller, R., Gabet, Y., Bab, I., Zilberman, U., Smith, P., 2004. New approach

to quantifying developmental variation in the dentition using serial microtomographic imaging. Microsc. Res. Tech., 65, 263-299.

Bailey, S.E., 2002. A closer look at Neanderthal postcanine dental morphology: the

mandibular dentition. Anat. Rec. (New Anat.) 269, 148-156. Bailey, S.E., 2006. Beyond shovel-shaped incisors: Neandertal dental morphology in a

comparative context. Periodicum Biologorum 108, 253-267. Bailey, S.E., Lynch, J.M., 2005. Diagnostic differences in mandibular P4 shape between

Neandertals and anatomically modern humans. Am. J. Phys. Anthropol. 126, 268-277.

Bailey, S.E., Hublin, J-J., 2006. Dental remains from the Grotte du Renne at Arcy-sur-

Cure (Yonne). J. Hum. Evol. 50, 485-508. Bailey, S.E., Wood, B.A., 2007. Trends in postcanine occlusal morphology within the

hominin clade: the case of Paranthropus. In: Bailey, S.E., Hublin, J.-J. (Eds.), Dental Perspectives on Human Evolution. Springer, Dordrecht, The Netherlands, pp. 33-52.

Becquet, C., Patterson, N., Stone, A. C., Przeworski, M., Reich, D., 2007. Genetic

structure of chimpanzee populations. PLos Genet. 3(4), e66. doi:10.1371/journal.pgen.0030066.

Bermúdez de Castro, J.M., Martínez, I., 1986. Hypocone and metaconule: identification

and variability on human molars. Int. J. Anthropol. 1, 165-168. Beynon, A.D., Dean, M.C., Reid, D.J., 1991. On thick and thin enamel in hominoids.

Am. J. Phys. Anthropol. 86, 295-309. Bookstein, F.L., 1991. Morphometric Tools for Landmark Data: Geometry and Biology.

Cambridge Univeristy Press, Cambridge. Bookstein, F.L., 1997. Landmark methods for forms without landmarks: morphometrics

of group differences in outline shape. Med. Image Anal. 1, 225-243. Butler, P.M., 1956. The ontogeny of molar pattern. Biological Reviews 31, 30-70. Butler, P.M., 1999. The relation of cusp development and calcification to growth. In:

Mayhall, J.T., Heikkinen, T. (Eds.), Proceedings of the 11th International Symposium on Dental Morphology, Oulu, Finland, 1998. Oulu University Press, Oulu, Finland, pp. 26-32.

168

Cai, J., Cho, S.-W., Kim, J.-Y., Lee, M.-J., Cha, Y.-G., Jung, H.-S., 2007. Patterning the size and number of tooth and its cusps. Dev. Biol. 304, 499-507.

Chai, Y., Jiang, X., Ito, Y., Bringas Jr., P., Han, J., Rowitch, D.H., Soriano, P.,

McMahon, A.P. and Sucov, H.M., 2000. Fate of mammalian neural crest during tooth and mandibular morphogenesis. Development 127, 1671-1679.

Cobourne, M.T., Sharpe, P.T., 2003. Tooth and jaw: molecular mechanisms of patterning

in the first branchial arch. Arch. Oral Biol. 48, 1-14. Conroy, G.C., 1991. Enamel thickness in South African australopithecines: noninvasive

evaluation by computed tomography. Palaeont. Afr. 28, 53-58. Conroy, G.C., Lichtman, J.W., Martin, L.B., 1995. Brief communication: some

obervations on enamel thickness and enamel prism packing in the Miocene hominoid Otavipithecus namibiensis. Am. J. Phys. Anthropol. 98, 595-600.

Coolidge, H.J., 1933. Pan paniscus. Pygmy chimpanzee from south of the Congo River.

Am. J. Phys. Anthropol. 18, 1-57. Corruccini, R.S., 1987a. The dentinoenamel junction in primates. Int. J. Primatol. 8, 99-

114. Corruccini, R.S., 1987b. Relative growth from the dentino-enamel junction in primate

maxillary molars. J. Hum. Evol. 2, 263-269. Corruccini, R.S., 1998. The dentino-enamel junction in primate mandibular molars. In:

Lukacs, J.R. (Ed.), Human Dental Development, Morphology, and Pathology: A Tribute to Albert A. Dahlberg. University of Oregon Anthropological Papers, Portland, pp. 1-16.

Corruccini, R.S., Holt, B.M., 1989. The dentinoenamel junction and the hypocone in

primates. Hum. Evol. 4, 253-262. Dahlberg, A.A., 1950. The evolutionary significance of the protostylid. Am. J. Phys.

Anthropol. 8, 15-25. Dassule, H.R., Lewis, P., Bei, M., Maas, R., McMahon, A.P., 2000. Sonic hedgehog

regulates growth and morphogenesis of the tooth. Development 127, 4775-4785. Donoghue, P.C.J., 2002. Evolution of development of the vertebrate dermal and oral

skeletons: unraveling concepts, regulatory theories, and homologies. Paleobiology 28, 474-504.

Dryden, I., Mardia, K.V., 1998. Statistical Shape Analysis. John Wiley & Sons, New

York.

169

Ferguson, C., Hardcastle, Z., Sharpe, P., 2000a. Development and patterning of the

dentition. In: Development, Growth and Evolution, O’Higgins, P., Cohn, M. (Eds.) Academic Press, London, pp. 188-205.

Ferguson, C.A., Tucker, A.S., Sharpe, P.T., 2000b. Temporospatial cell interactions

regulating mandibular and maxillary arch patterning. Development 127, 403-412. Fischer, A., Pollack, J., Thalmann, O., Nickel, B., Pääbo, S., 2006. Demographic history

and genetic differentiation in apes. Curr. Biol. 16, 1133-1138. Grine, F.E., 1989. New hominid fossils from the Swartkrans formation (1979-1986

excavations): craniodental specimens. Amer. J. Phys. Anthropol. 79, 409-449. Grine, F.E., 1991. Computed tomography and the measurement of enamel thickness in

extant hominoids: implications for its palaeontological application. Palaeont. Afr. 28, 61-69.

Grine, F.E., 2004. Description and preliminary analysis of new hominid craniodental

remains from the Swartkrans Formation. In: Brain, C.K. (Ed.), A Cave’s Chronicle of Early Man. Transvaal Museum, Pretoria, pp. 75-116.

Grine, F.E., Martin, L.B., 1988. Enamel thickness and development in Australopithecus

and Paranthropus. In: Grine, F.E. (Ed.), Evolutionary History of the “Robust” Australopithecines. Aldine de Gruyter, New York, pp. 3-42.

Gonder, M.K., Disotell, T.R., Oates, J.F., 2006. New genetic evidence on the evolution of

chimpanzee populations and implications for taxonomy. Int. J. Prim. 27, 1103-1127. Gower, J.C., 1975. Generalized Procrustes analysis. Psychometrika 40, 33-51. Guatelli-Steinberg, D., Irish, J.D., 2005. Early hominin variability in first molar dental

trait frequencies. Am. J. Phys. Anthropol. 128, 477-484. Gunz, P., Harvati, K., 2007. The Neanderthal “chignon”: variation, integration, and

homology. J. Hum. Evol. 52, 262-274. Gunz, P., Mitteroecker, P., Bookstein, F.L., 2005. Semilandmarks in three dimensions.

In: Slice, D.E. (Ed.), Modern Morphometrics in Physical Anthropology. Kluwer Academic/Plenum Publishers, New York, pp. 73-98.

Hammer, O., Harper, D., 2006. Paleontological Data Analysis. Blackwell Publishing,

Oxford. Harris, E.F., Dinh, D.P., 2006. Intercusp relationships of the permanent maxillary first

and second molars in American whites. Am. J. Phys. Anthropol. 130, 514-528.

170

Hartman, S.E., 1988. A cladistic analysis of hominoid molars. J. Hum. Evol. 17, 489-502. Hlusko, L.J., 2002. Expression types for two cercopithecoid dental traits (interconulus

and interconulid) and their variation in a modern baboon population. Int. J. Primatol. 23, 1309-1318.

Hlusko, L.J., 2004. Protostylid variation in Australopithecus. J. Hum. Evol. 46, 579-594. Hlusko, L.J., Mahaney, M.C., 2003. Genetic contributions to expression of the baboon

cingular remnant. Arch. Oral Biol. 48, 663-672. Irish, J., Gautelli-Steinberg, D., 2003. Ancient teeth and modern human origins: an

expanded comparison of African Plio-Pleistocene and recent world dental samples. J. Hum. Evol. 45, 113-144.

Jernvall, J., Åberg, T., Kettunen, P., Keränen, S., Thesleff, I., 1998. The life history of an

embryonic signaling center: BMP-4 induces p21 and is associated with apoptosis in the mouse tooth enamel knot. Development 125, 161-169.

Jernvall, J., 2000. Linking development with generation of novelty in mammalian teeth.

Proc. Nat. Acad. Sci. U.S.A. 97, 2641-2645. Jernvall, J., Jung, H.-S., 2000. Genotype, phenotype, and developmental biology of molar

tooth characters. Year. Phys. Anthropol. 43, 171-190. Jernvall, J., Keranen, S., Thesleff, I., 2000. Evolutionary modification of development in

mammalian teeth: quantifying gene expression patterns and topography. Proc. Nat. Acad. Sci. U.S.A. 97, 14444-14448.

Jernvall, J.,Thesleff, I., 2000. Reiterative signalling and patterning during mammalian

tooth morphogenesis. Mech. Dev. 92, 19-29. Johanson, D.C., 1974. An odontological study of the chimpanzee with some implications

for hominoid evolution. Ph.D. Dissertation, University of Chicago. Jungers, W.L., Falsetti, A.B., Wall, C.E., 1995. Shape, relative size and size-adjustment

in morphometrics. Yearb. Phys. Anthropol. 38, 137-161. Kangas, A.T., Evans, A.R., Thesleff, I., Jernvall, J., 2004. Nonindependence of

mammalian dental characters. Nature 432, 211-214. Kassai, Y., Munne, P., Hotta, Y., Penttilä, E., Kavanagh, K., Ohbayashi, N., Takada, S.,

Thesleff, I., Jernvall, J., Itoh, N., 2005. Regulation of mammalian tooth cusp patterning by ectodin. Science 309, 2067-2070.

171

Kavanagh, K., Evans, A., Jernvall, J., 2007. Predicting evolutionary patterns of mammalian teeth from development. Nature 499, 427-432.

Keyser, A.W., Menter, C.G., Moggi-Cecchi, J., Pickering, T.R., Berger, L.R., 2000.

Drimolen: a new hominid-bearing site in Gauteng, South Africa. S. Afr. J. Sci. 96, 193-197.

King, S.J., Arrigo-Nelson, S.J., Pochron, S.T., Semprebon, G.M., Godfrey, L.R., Wright,

P.C., Jernvall, J., 2005. Dental senescence in a long-lived primate links infant survival to rainfall. Proc. Nat. Acad. Sci., U.S.A. 102, 16579-16583.

Kono, R.T., 2004. Molar enamel thickness and distribution patterns in extant great apes

and humans: new insights based on a 3-dimensional whole crown perspective. Anthropol. Sci. 112, 121-146.

Kono, R.T., Suwa, G., Tanijiri, T., 2000. Three-dimensional reconstruction and analysis

of serially scanned CT image data using CT-Rugle software. Gazolabo 11, 48-52 (in Japanese).

Kono R.T., Suwa, G., Tanijiri, T., 2002. A three-dimensional analysis of enamel

distribution patterns in human permanent first molars. Arch. Oral Biol. 47, 867-875. Korenhof, C.A.W., 1960. Morphogenetical Aspects of the Human Upper Molar.

Uitgeversmaatschappij Neerlandia, Utrecht. Korenhof, C.A.W., 1961. The enamel-dentine border: a new morphological factor in the

study of the (human) molar pattern. Proc. Koninkl., Nederl. Acad. Wetensch. 64B, 639-664.

Korenhof, C.A.W., 1978. De evolutie van het ondermolaarpatroon en overblijfselen van

het trigonid bij de mens (I). Ned. Tijdschr. Tandheelkd. 85, 456-495. Korenhof, C.A.W., 1982. Evolutionary trends of the inner enamel anatomy of deciduous

molars from Sangiran (Java, Indonesia). In: Kurtén, B. (Ed.), Teeth: Form, Function and Evolution. Columbia University Press, New York, pp. 350-365.

Kortlandt A., 1995. A survey of the geographical range, habitats and conservation of the

pygmy chimpanzee (Pan paniscus): an ecological perspective. Prim. Cons. 16: 21-36. Kraus, B.S., 1952. Morphologic relationships between enamel and dentin surfaces of

lower first molar teeth. J. Dent. Res. 31, 248-256. Kraus, B.S., Jordan, R., 1965. The Human Dentition Before Birth. Lea and Febiger,

Philadelphia.

172

Kraus, B.S., Oka, S.W., 1967. Wrinkling of molar crowns: new evidence. Science 157, 328-329.

Kunimatsu, Y., Nakatsukasa, M., Sawada, Y., Sakai, T., Hyodo, M., Hyodo, H., Itaya, T.,

Nakaya, H., Saegusa, H., Mazurier, A., Saneyoshi, M., Tsujikawa, H., Yamamoto, A., Mbua, E., 2007. A new Late Miocene great ape from Kenya and its implications for the origins of African great apes and humans. Proc. Nat. Acad. Sci. U.S.A. 104, 19220-19225.

Line, S.R.P., 2003. Variation of tooth number in mammalian dentition: connecting

genetics, development and evolution. Evol. Dev. 5, 295-304. Lumsden, A.G., 1988. Spatial organization of the epithelium and the role of neural crest

cells in the initiation of the mammalian tooth germ. Development 103, 155-169. Macchiarelli, R., Bondioli, L., Debénath, A., Mazurier, A., Tournepiche, J.-F., Birch,W.,

Dean, C., 2006. How Neanderthal molar teeth grew. Nature 444, 748-751. Macho, G.A., Thackeray, J.F., 1992. Computed tomography and enamel thickness of

maxillary molars of Plio-Pleistocene hominids from Sterkfontein, Swartkrans, and Kromdraai, (South Africa): and exploratory study. Am. J. Phys. Anthropol. 89, 133-143.

Martinón-Torres, M., Bermúdez de Castro, J.M., Gómez-Robles, A., Arsuaga, J.L.,

Carbonell, E., Lordkipanidze, D., Manzi, G., Margvelashvili, A., 2007. Dental evidence on the hominin dispersals during the Pleistocene. Proc. Natl. Acad. Sci. U.S.A. 104, 13279-13282.

McCollum, M.A., Sharpe, P., 2001. Developmental genetics and early hominid

craniodental evolution. BioEssays 23, 481-493. Menter, C.G., Kuykendall, K.L., Keyser, A.W., Conroy, G.C., 1999. First record of

hominid teeth from the Plio-Pleistocene site of Gondolin, South Africa. J. Hum. Evol. 37, 299-307.

Mina, M., Kollar, E.J., 1987. The induction of odontogenesis in non-dental mesenchyme

combined with early murine mandibular arch epithelium. Arch. Oral Biol. 32, 123-127.

Moggi-Cecchi, J., Grine, F.E., Tobias, P.T., 2006. Early hominid dental remains from

Members 4 and 5 of the Sterkfontein Formation (1966-1996 excavations): catalogue, individual associations, morphological descriptions and initial metric analysis. J. Hum. Evol. 50, 239-328.

173

Morin, P.A., Moore, J.J., Chakraborty, R., Jin, L., Goodall, J., Woodruff, D.S., 1994. Kin selection, social structure, gene flow, and the evolution of chimpanzees. Science 265: 1193-1201.

Mosimann, J.E., 1970. Size allometry: size, and shape variables with characteristics of

the log normal and generalized gamma distributions. J. Am. Stat. Assoc. 655, 930-945.

Nager, G., 1960. Der vergleich zwischen dem räumlichen verhalten des dentin-

kronenreliefs und dem schmelzrelief der zahnkrone. Acta Anat. 42, 226-250. Neubuser, A., Peters, H., Balling, R., Martin, G.R., 1997. Antagonistic interactions

between FGF and BMP signaling pathways: a mechanism for positioning the sites of tooth formation. Cell 90, 247-255.

Olejniczak, A.J., 2006. Micro-computed tomography of primate molars. Ph.D.

Dissertation, Stony Brook University. Olejniczak, A.J., Gilbert, C.C., Martin, L.B., Smith, T.M., Ulhaas, L., Grine, F.E., 2007.

Morphology of the enamel-dentine junction in sections of anthropoid primate maxillary molars. J. Hum. Evol. 53, 292-301.

Olejniczak, A.J., Grine, F.E., 2005. High-resolution measurement of Neandertal tooth

enamel thickness by micro-focal computed tomography. S. Afr. J. Sci. 101, 219-220. Olejniczak, A.J., Grine, F.E., 2006. Assessment of the accuracy of dental enamel

thickness measurements using micro-focal X-ray computed tomography. Anatomical Record A 288, 263-275.

Olejniczak, A.J., Martin, L.B., Ulhaas, L., 2004. Quantification of dentine shape in

anthropoid primates. Ann. Anat. 186, 479-485. Olejniczak, A.J., Smith, T.M., Wang, W., Potts, R., Ciochon, R., Kullmer, O., Schrenk,

F., Hublin, J-J., 2008. Molar enamel thickness and dentine horn height in Gigantopithecus blacki. Am. J. Phys. Anthropol. 135, 85-91.

Olejniczak A.J., Smith T.M., Skinner M.M., Grine F.E., Feeney R.N.M., Thackeray J.F.,

Hublin, J.-J., n.d. Three-dimensional molar enamel distribution and thickness in Australopithecus and Paranthropus. Biology Letters.

Osborn, J.W., 1978. Morphogenetic gradients: fields versus clones. In: Development,

Function and Evolution of Teeth, Butler, P.M., Joysey, K.A. (Eds.). Academic Press London, pp. 171-201.

174

Pilbrow, V., 2003. Dental variation in African apes with implications for understanding patterns of variation in species of fossil apes. Ph.D. Dissertation, New York University.

Pilbrow, V., 2006a. Population systematics of chimpanzees using molar morphometrics.

J. Hum. Evol. 51, 646-662. Pilbrow, V., 2006b. Lingual incisor traits in modern hominoids and an assessment of

their utility for fossil hominoid taxonomy. Am. J. Phys. Anthropol. 129, 323-338. Polly, P.D., 1998. Variability, selection, and constraints: development and evolution in

viverravid (Carnivora, Mammalia) molar morphology. Paleobiology 24, 409-429. Reid, C., Van Reenen, J., 1995. The Carabelli trait in early South African hominids: a

morphometric study. In: Moggi-Cecchi, J. (Ed.), Aspects of Dental Biology: Paleontology, Anthropology and Evolution. International Institute for the Study of Man, Florence, pp. 299-304.

Robinson, J.T., 1956. The Dentition of the Australopithecinae. Transvaal Museum,

Pretoria. Robinson, J.T., 1963. Adaptive radiation in the australopithecines and the origin of man.

In: Clark Howell, F., Bourliere, F. (Eds.), African Ecology and Human Evolution. Aldine: Chicago, pp. 385-416.

Rohlf, F.J., 1993. Relative warp analysis and an example of its application to mosquito

wings. In: Marcus, L.F., Bello, E., Garcia-Valdecasas, A. (Eds.), Contributions to Morphometrics. Msueo Nacional de Ciencias Naturales, Madrid, pp. 131-159.

Rohlf, F.J., Slice, D., 1990. Extensions of the Procrustes method for the optimal

superimposition of landmarks. Syst. Zool. 39, 40-59. Rosas, A., Martínez-Maza, C., Bastir, M., García-Tabernero, A., Lalueza-Fox, C.,

Huguet, R., Ortiz, J.E., Julià, R., Soler, V., de Torres, T., Martínez, E., Cañaveras, J.C., Sánchez-Moral, S, Cuezva, S., Lario, J., Santamaría, D., de la Rasilla, M., Fortea, J., 2006. Paleobiology and comparative morphology of a late Neandertal sample from El Sidron, Asturias, Spain. Proc. Nat. Acad. Sci. U. S. A. 103, 19266-19271.

Ruvolo, M., Pan, D., Zehr, S., Goldberg, T., Disotell, T.R., von Dornum, M., 1994. Gene

trees and hominoid phylogeny. Proc. Nat. Acad. Sci., U.S.A. 91, 8900-8904. Sakai, T., Hanamura, H., 1971. A morphology study of enamel-dentin border on the

Japanese dentition. Part V. Maxillary molar. J. Anthropol. Soc. Nippon 79, 297-322.

175

Sakai, T., Hanamura, H., 1973a. A morphology study of enamel-dentin border on the Japanese dentition. Part VI. Mandibular molar. J. Anthropol. Soc. Nippon 81, 25-45.

Sakai, T., Hanamura, H., 1973b. A morphology study of enamel-dentin border on the

Japanese dentition. Part VII. General conclusion. J. Anthropol. Soc. Nippon 81, 87-102.

Sakai, T., Sasaki, I., Hanamura, H., 1965. A morphology study of enamel-dentin border

on the Japanese dentition. Part I. Maxillary median incisor. J. Anthropol. Soc. Nippon 73, 91-109.

Sakai, T., Sasaki, I., Hanamura, H., 1967a. A morphology study of enamel-dentin border

on the Japanese dentition. Part II. Maxillary canine. J. Anthropol. Soc. Nippon 75, 155-172.

Sakai, T., Sasaki, I., Hanamura, H., 1967b. A morphology study of enamel-dentin border

on the Japanese dentition. Part III. Maxillary premolar. J. Anthropol. Soc. Nippon 75, 207-223.

Sakai, T., Sasaki, I., Hanamura, H., 1969. A morphology study of enamel-dentin border

on the Japanese dentition. Part IV. Mandibular premolar. J. Anthropol. Soc. Nippon 77, 71-98.

Salazar-Ciudad, I., Jernvall, J., Newman, S., 2003. Mechanisms of pattern formation in development and evolution. Development 130, 2027-2037.

Sasaki, K., Kanazawa, E., 1999. Morphological traits on the dentino-ename junction of

lower deciduous molar series. In: Mayhall, J.T., Heikkinen, T. (Eds.), Proceedings of the 11th International Symposium on Dental Morphology, Oulu, Finland, 1998. Oulu University Press, Oulu, Finland, pp. 167-178.

Satokata, I., Maas, R., 1994. Msx1 deficient mice exhibit cleft palate and abnormalities of

craniofacial and tooth development. Nat. Genet. 6, 348-356. Schulze, M.A., Pearce, J.A., 1994. A morphology-based filter structure for edge-

enhancing smoothing. In Proceedings of the 1994 IEEE International Conference on Image Processing, pp. 530-534.

Scott, G.R., Turner II, C.G., 1997. The Anthropology of Modern Human Teeth.

Cambridge University Press, Cambridge. Schwartz, G.T., Thackeray, J.F., Reid, C., van Reenan, J.F., 1998. Enamel thickness and

the topography of the enamel-dentine junction in South African Plio-Pleistocene hominids with special reference to the Carabelli trait. J. Hum. Evol. 35, 523-542.

Sharpe, P.T., 1995. Homeobox genes and orofacial development. Con. Tiss. Res. 32, 17-

25.

176

Shea, B.T., Leigh, S.R., Groves, C.P., 1993. Mulivariate craniometric variation in

chimpanzees: implications for species identification in paleoanthropology. In: Kimbel, W.H., Martin, L.B. (Eds.), Species, Species Concepts and Primate Evolution. Plenum Press, New York, pp. 265-296.

Shimizu, D., 2002. Functional implications of enamel thickness in the lower molars of

red colobus (Procolobus badius) and Japanese macaque (Macaca fuscata). J. Hum. Evol. 43, 605-620.

Scott, G.R., Turner II, C.G., 1997. The Anthropology of Modern Human Teeth.

Cambridge University Press, Cambridge. Skinner, M.M. Gunz, P., Wood, B.A., Hublin, J-J., n.d. Enamel-dentine junction

morphology distinguishes the lower molars of Australopithecus africanus and Paranthropus robustus. J. Hum. Evol.

Skinner, M.M, Wood, B.A., Boesch, C., Olejniczak, A.J., Rosas, A., Smith, T.M.,

Hublin, J-J., 2008. Dental trait expression at the enamel-dentine junction of lower molars in extant and fossil hominoids. J. Hum. Evol. doi:10.1016/j.jhevol.2007.09.012.

Smith, P., Gomorri, J.M., Spitz, S., Becker, J., 1997. Model for the examination of

evolutionary trends in tooth development. Am. J. Phys. Anthropol. 102, 283-294. Smith, T.M., Olejniczak, A.J., Reid, D.J., Ferrell, R., Hublin, J-J., 2006a. Modern human

molar enamel thickness and enamel dentine junction shape. Arch. Oral Biol. 51, 974-995.

Smith, T.M., Olejniczak, A.J., Tafforeau, P., Reid, D.J., Grine, F.E., Hublin, J-J., 2006b.

Molar crown thickness, volume, and development in South African Middle Stone Age humans. S. Afr. J. Sci. 102, 513-517.

Sperber, G., 1974. Morphology of the cheek teeth of early South African hominids. Ph.D.

Dissertation, University of Witwatersrand. Stone, A.C., Griffiths, R.C., Zegura, S.L., Hammer, M.F., 2002. High levels of Y-

chromosome nucleotide diversity in the genus Pan. Proc. Nat. Acad. Sci., U.S.A. 99, 43-48.

Suwa, G., 1996. Serial allocations of isolated mandibular molars of unknown taxonomic

affinities from the Shungura and Unso Formations, Ethiopia, a combined method approach. Human Evolution 11, 269-282.

177

Suwa, G. Kono, R.T., 2005. A micro-CT based study of linear enamel thickness in the mesial cusp section of human molars: reevaluation of methodology and assessment of within-tooth, serial, and individual variation. Anthropol. Sci. 113, 273-289.

Suwa, G., Kono, R.T., Katoh, S., Asfaw, B., Beyene, Y., 2007. A new species of great

ape from the late Miocene epoch in Ethiopia. Nature 448, 921-924. Suwa, G., White, T.D., Howell, F.C., 1996. Mandibular postcanine dentition from the

Shungura Formation, Ethiopia: crown morphology, taxonomic allocations, and Plio-Pleistocene hominid evolution. Am. J. Phys. Anthropol. 101, 247-282.

Suwa, G., Wood, B.A., White, T.D., 1994. Further analysis of mandibular molar crown

and cusp areas in Pliocene and Early Pleistocene hominids. American Journal of Physical Anthropology 93, 407-426.

Swails, N.J., 1993. The evolutionary implications of primate tooth-germ development:

using ontogenetic data to make phylogenetic inferences. Ph.D. Dissertation, University of Washington.

Tafforeau, P., 2004. Phylogenetic and functional aspects of tooth enamel microstructure

and three-dimensional structure of modern and fossil primate molars. Ph.D. Dissertation, Université de Montpellier II.

Ten Cate, A.R. (2007). Oral Histology: Development, Structure and Function, 7th

Edition. C.V. Mosby Company, St. Louis. Thomas, B.L., Tucker, A.S., Qiu, M., Ferguson, C., Hardcastle, Z., Rubenstein, J.L.R.,

Sharpe, P.T., 1997. Role of Dlx-1 and Dlx-2 genes in patterning of the murine dentition. Development 124, 4811-4818.

Tobias, P.V., Copley, K., Brain, C., 1977. South Africa. In: Catalogue of Fossil

Hominids. Part 1: Africa (2nd Edition), Oakley, K.P., Campbell, B.G., Molleson, T. I., (Eds.). British Museum of Natural History, London.

Tucker, A.S., Headon, D.J., Courtney, J.-M., Overbeek, P., Sharpe, P.T., 2004. The

activation level of the TNF family receptor, Edar, determines cusp number and tooth number during tooth development. Dev. Biol. 268, 185-194.

Tucker, A.S., Matthews, K.L., Sharpe, P.T., 1998. Transformation of tooth type induced

by inhibition of BMP signaling. Science 282, 1136-1138. Turner II, C.G., Nichol, C.R., Scott, G.R., 1991. Scoring procedures for key

morphological traits of the permanent dentition: the Arizona State University Dental Anthropology System. In: Kelley, M.A., Larsen, C.S. (Eds.), Advances in Dental Anthropology. Wiley-Liss, New York, pp. 13-31.

178

Uchida, A., 1992. Intra-species variation among the great apes: implications for taxonomy of fossil hominoids. Ph.D. Dissertation, Harvard University.

Uchida, A., 1996. Craniodental Variation Among the Great Apes. Peabody Museum of

Archaeology and Ethnology, Harvard University, Cambridge. Uchida, A., 1998a. Variation in tooth morphology of Gorilla gorilla. J. Hum. Evol. 34,

55-70. Uchida, A., 1998b. Variation in tooth morphology of Pongo pygmaeus. J. Hum. Evol. 34,

71-79. van Genderen, C., Okamura, R.M., Farinas, I., 1994. Development of several organs that

require inductive epithelial-mesenchymal interactions is impaired in LEF-1 deficient mice. Gen. Dev. 8, 2691-2703.

Van Reenen, J., Reid, C., 1995. The Carabelli trait in early South African hominids: a

morphological study. In: Moggi-Cecchi, J. (Ed.), Aspects of Dental Biology: Paleontology, Anthropology and Evolution. International Institute for the Study of Man, Florence, pp. 291-298.

von Koenigswald, G.H.R., 1952. Gigantopithecus blacki, a giant fossil hominid from the

Pleistocene of southern China. Anthropol. Pap. Am. Mus. Nat. Hist. 43, 295-325. Weaver, T.D., Roseman, C.C., Stringer, C.B., 2007. Were Neandertal and modern human

cranial differences produced by natural selection or genetic drift. J. Hum. Evol. 53, 135-145.

Weidenreich, F., 1937. The dentition of Sinanthropus pekinensis: a comparative

odontography of the hominids. Palaeontologica Sinica, Series D, I, 1-180. Weidenreich, F., 1945. Giant early man rom Java and South China. Anthropol. Pap. Am.

Mus. Nat. Hist. 40, 1-134. Weiss, K.M., Stock, D.W., Zhao, Z., 1998. Dynamic interactions and the evolutionary

genetics of dental patterning. Crit. Rev. Oral Biol. Med. 9, 369-398. Won, Y-J., Hey, J., 2005. Divergence population genetics of chimpanzees. Mol. Biol.

Evol. 22, 297-307. Wood, B.A., 1991. Hominid Cranial Remains. In: Koobi Fora Research Project, vol. 4.

Clarendon Press, Oxford. Wood, B.A., Abbott, S.A., 1983. Analysis of the dental morphology of Plio-Pleistocene

hominids. I. Mandibular molars: crown area measurements and morphological traits. J. Anat. 136, 197-219.

179

Wood, B.A., Abbott, S.A., Graham, S.H., 1983. Analysis of the dental morphology of

Plio-Plesitocene hominids. II. Mandibular molars - study of cusp areas, fissure patterns and cross sectional shape of the crown. J. Anat. 137, 287-314.

Wood, B.A., Constantino, P., 2007. Paranthropus boisei: fifty years of evidence and

analysis. Yearb. Phys. Anthropol. 50, 106-132. Wood, B.A., Richmond, B.R., 2000. Human evolution: taxonomy and paleobiology. J.

Anat. 196, 19-60. Wu, L., Turner, C.G., 1993. Variation in the frequency and form of the lower permanent

molar middle trigonid crest. Am. J. Phys. Anthropol. 91, 245-248. Zilberman, U., Skinner, M., Smith, P., 1992. Tooth components of mandibular deciduous

molars of Homo sapiens sapiens and Homo sapiens neanderthalensis: a radiographic study. Am. J. Phys. Anthropol. 87, 255-262.

180

Appendix A. Molar sample used in this thesis including locality/site information and inclusion in particular analyses.

Genus Species Subspecies Source1 Locality or Site Accession2 Tooth Side Chap_2 Chap_3 Chap_4 Chap_5

Gorilla gorilla beringei NMNH Rwanda 545037 LM2 R Y Y

Gorilla gorilla beringei NMNH Rwanda 543034 LM3 R Y Y

Homo sapiens sapiens NMNH Unknown SI_34* LM1/2 L Y

Homo sapiens sapiens NMNH Unknown SI_37* LM1/2 L Y

Homo sapiens sapiens MPI-EVA Inverness, Scotland SKB_30* LM2 L Y

Homo sapiens sapiens MPI-EVA Garrowby Wold 101 SKB_71* LM2 R Y

Pan paniscus paniscus MRAC DR Congo, Ibembo 5374 LM1 L Y

Pan paniscus paniscus MRAC DR Congo, Ponthierville 27009 LM1 L Y

Pan paniscus paniscus MRAC DR Congo, Djolu 29010 LM1 R Y

Pan paniscus paniscus MRAC DR Congo, Djolu 29016 LM1 R Y

Pan paniscus paniscus MRAC DR Congo, Djolu 29024 LM1 L Y

Pan paniscus paniscus MRAC DR Congo, Djolu 29026 LM1 R Y

Pan paniscus paniscus MRAC DR Congo, Djolu 29030 LM1 R Y

Pan paniscus paniscus MRAC DR Congo, Dongo 29048 LM1 R Y

Pan sp ssp MRAC Unknown 84036M11 LM1 L Y

Pan paniscus paniscus MRAC DR Congo, Coquilhatville 22908 LM2 L Y

Pan paniscus paniscus MRAC DR Congo, Djolu 29030 LM2 L Y

Pan paniscus paniscus MRAC DR Congo, Djolu 29033 LM2 L Y

Pan paniscus paniscus MRAC DR Congo, Bokungu 29041 LM2 L Y

Pan paniscus paniscus MRAC DR Congo, Batiakayandja 29055 LM2 L Y

Pan paniscus paniscus MRAC DR Congo, Bore 29056 LM2 R Y

Pan paniscus paniscus MRAC DR Congo, Lubutu 29066 LM2 L Y

Pan paniscus paniscus MRAC DR Congo, Ponthierville 84036M03 LM2 R Y

Pan paniscus paniscus MRAC DR Congo, Yahuma 84036M04 LM2 L Y

Pan sp ssp MRAC Unknown 84036M10 LM2 L Y

Pan sp ssp MRAC Unknown 84036M11 LM2 L Y

Pongo pygmaeus abelli ZMB Sumatra, Indonesia 38606 LM1 L Y

Pongo pygmaeus pygmaeus ZMB Borneo, Indonesia 30946 LM1 R Y

181

Pongo pygmaeus ssp ZMB Captive 6987 LM1 L Y

Pan sp ssp ZMB Unknown 20811 LM1 R Y

Pan sp ssp ZMB Captive 47506 LM1 L Y

Pan sp ssp ZMB Captive 72844 LM1 R Y

Pan sp ssp ZMB Unknown 0.A809 LM1 R Y

Pan troglodytes troglodytes ZMB Cameroon, Ebolowa 15849 LM1 L Y

Pan troglodytes troglodytes ZMB Cameroon, Akonolinga 30847 LM1 R Y

Pan sp ssp ZMB Unknown 32052 LM1 R Y

Pan sp ssp ZMB Unknown 32356 LM1 R Y

Pan troglodytes troglodytes ZMB Cameroon, Yaounde 35526 LM1 R Y

Pan troglodytes troglodytes ZMB Cameroon, Bipindi 83604 LM1 L Y

Pan troglodytes troglodytes ZMB Cameroon, Yakuduma 83623 LM1 L Y

Pan troglodytes troglodytes ZMB Cameroon, Ukalla Nsama 83673 LM1 L Y

Pan troglodytes troglodytes ZMB Cameroon, Basho A162.07 LM1 L Y

Pan troglodytes verus MPI-EVA Cote D’Ivoire, Tai Forest 11791 LM1 L Y

Pan troglodytes verus MPI-EVA Cote D’Ivoire, Tai Forest 11792 LM1 R Y

Pan troglodytes verus MPI-EVA Cote D’Ivoire, Tai Forest 11798 LM1 L Y Y

Pan troglodytes verus MPI-EVA Cote D’Ivoire, Tai Forest 11800 LM1 R Y

Pan troglodytes verus MPI-EVA Cote D’Ivoire, Tai Forest 11903 LM1 R Y

Pan troglodytes verus MPI-EVA Cote D’Ivoire, Tai Forest 12176 LM1 R Y

Pan troglodytes verus MPI-EVA Cote D’Ivoire, Tai Forest 13433 LM1 L Y Y

Pan troglodytes verus MPI-EVA Cote D’Ivoire, Tai Forest 14992 LM1 L Y Y

Pan troglodytes verus MPI-EVA Cote D’Ivoire, Tai Forest 14995 LM1 L Y Y

Pan troglodytes verus MPI-EVA Cote D’Ivoire, Tai Forest 15011 LM1 R Y

Pan sp ssp ZMB West Africa 6983 LM1 R Y

Pan sp ssp ZMB Bugoie Wald, Rwanda 24838 LM2 L Y

Pan sp ssp ZMB Egypt 33489 LM2 L Y

Pan sp ssp ZMB Captive 72844 LM2 R Y

Pan troglodytes troglodytes ZMB Cameroon, Akonolinga 30846 LM2 R Y

Pan troglodytes troglodytes ZMB Cameroon, Akonolinga 30847 LM2 L Y

182

Pan troglodytes troglodytes ZMB Mburu Ogowe (Gabon?) 31279 LM2 R Y

Pan troglodytes troglodytes ZMB Cameroon, Buea 46095 LM2 L Y

Pan troglodytes troglodytes ZMB Cameroon, Lomie 83635 LM2 L Y

Pan troglodytes troglodytes ZMB Cameroon, Akonolinga 83639 LM2 R Y

Pan troglodytes troglodytes ZMB Gabon, Mayumba 83685 LM2 R Y

Pan troglodytes verus MPI-EVA Côte d'Ivoire, Tai Forest 11778 LM2 R Y

Pan troglodytes verus MPI-EVA Côte d'Ivoire, Tai Forest 11779 LM2 R Y

Pan troglodytes verus MPI-EVA Côte d'Ivoire, Tai Forest 11784 LM2 R Y

Pan troglodytes verus MPI-EVA Côte d'Ivoire, Tai Forest 11790 LM2 R Y Y

Pan troglodytes verus MPI-EVA Côte d'Ivoire, Tai Forest 11791 LM2 R Y

Pan troglodytes verus MPI-EVA Côte d'Ivoire, Tai Forest 11792 LM2 L Y

Pan troglodytes verus MPI-EVA Côte d'Ivoire, Tai Forest 11800 LM2 R Y Y

Pan troglodytes verus MPI-EVA Côte d'Ivoire, Tai Forest 12176 LM2 R Y Y

Pan troglodytes verus MPI-EVA Côte d'Ivoire, Tai Forest 13433 LM2 R Y Y

Pan troglodytes verus MPI-EVA Côte d'Ivoire, Tai Forest 13437 LM2 R Y Y

Pan troglodytes verus MPI-EVA Côte d'Ivoire, Tai Forest 13438 LM2 R Y

Pan troglodytes verus MPI-EVA Côte d'Ivoire, Tai Forest 13439 LM2 R Y

Pan troglodytes verus MPI-EVA Côte d'Ivoire, Tai Forest 14992 LM2 L Y Y

Pan troglodytes verus MPI-EVA Côte d'Ivoire, Tai Forest 15008 LM2 R Y

Pan troglodytes verus MPI-EVA Côte d'Ivoire, Tai Forest 15012 LM2 R Y Y

Pan troglodytes verus MPI-EVA Côte d'Ivoire, Tai Forest 15014 LM2 R Y

Pan troglodytes verus MPI-EVA Côte d'Ivoire, Tai Forest 11790 LM3 L Y

Gigantopithecus blacki blacki SFN Unknown CA_736 LM2/3 L Y Y

Homo neanderthalensis neanderthalensis MNCN El Sidron, Spain SD_331c LM1 L Y

Homo neanderthalensis neanderthalensis MNCN El Sidron, Spain SD_540 LM1 L Y

Homo neanderthalensis neanderthalensis MNCN El Sidron, Spain SD_756 LM1 R Y

Homo neanderthalensis neanderthalensis MNCN El Sidron, Spain SD_780 LM1 L Y

Australopithecus africanus africanus UW Sterkfontein, South Africa STW_145 LM1 R Y Y

Australopithecus africanus africanus UW Sterkfontein, South Africa STW_291 LM1 R Y

Australopithecus africanus africanus UW Sterkfontein, South Africa STW_364 LM1 R Y

183

Australopithecus africanus africanus TM Sterkfontein, South Africa STS_9 LM1 R Y Y

Australopithecus africanus africanus UW Sterkfontein, South Africa STW_309A LM1 R Y Y

Australopithecus africanus africanus UW Sterkfontein, South Africa STW_421A LM1 R Y Y

Australopithecus africanus africanus UW Sterkfontein, South Africa STW_421B LM1 L Y Y

Australopithecus africanus africanus UW Sterkfontein, South Africa STW_3 LM2 L Y Y

Australopithecus africanus africanus UW Sterkfontein, South Africa STW_296 LM2 R Y Y

Australopithecus africanus africanus UW Sterkfontein, South Africa STW_424 LM2 L Y Y Y

Australopithecus africanus africanus UW Sterkfontein, South Africa STW_213 LM2 L Y Y

Australopithecus africanus africanus UW Sterkfontein, South Africa STW_213 LM2 R Y

Australopithecus africanus africanus UW Sterkfontein, South Africa STW_412A LM2 R Y

Australopithecus africanus africanus UW Sterkfontein, South Africa STW_412B LM2 L Y Y

Australopithecus africanus africanus UW Sterkfontein, South Africa STW_537 LM2 R Y Y

Australopithecus africanus africanus UW Sterkfontein, South Africa STW_537 LM2 L Y Y

Australopithecus africanus africanus UW Sterkfontein, South Africa STW_560D LM2 L Y

Australopithecus africanus africanus UW Sterkfontein, South Africa STW_560E LM2 R Y Y Y

Australopithecus africanus africanus TM Sterkfontein, South Africa TM_1520 LM3 L Y

Australopithecus africanus africanus UW Sterkfontein, South Africa STW_520 LM3 R Y Y

Australopithecus africanus africanus UW Sterkfontein, South Africa STS_55B LM3 L Y

Australopithecus africanus africanus UW Sterkfontein, South Africa STW_280 LM3 R Y Y

Australopithecus africanus africanus UW Sterkfontein, South Africa STW_491 LM3 L Y Y

Australopithecus africanus africanus UW Sterkfontein, South Africa STW_529 LM3 L Y Y

Australopithecus africanus africanus UW Sterkfontein, South Africa STW_537 LM3 L Y

Australopithecus africanus africanus UW Sterkfontein, South Africa STW_537 LM3 R Y Y

Australopithecus africanus africanus UW Sterkfontein, South Africa STW_560A LM3 R Y Y

Australopithecus africanus africanus UW Sterkfontein, South Africa STW_560B LM3 L Y Y

Paranthropus cf. robustus robustus UW Gondolin, South Africa GDA_2 LM2 L Y

Paranthropus robustus robustus TM Swartkrans, South Africa SK_104 LM1 R Y Y

Paranthropus robustus robustus TM Swartkrans, South Africa SK_3974 LM1 R Y Y Y

Paranthropus robustus robustus TM Swartkrans, South Africa SK_828 LM1 L Y Y Y

Paranthropus robustus robustus TM Swartkrans, South Africa SK_846A LM1 R Y

184

Paranthropus robustus robustus UW Drimolen, South Africa DNH_60b LM1 R Y Y

Paranthropus robustus robustus UW Drimolen, South Africa DNH_67 LM1 R Y Y Y

Paranthropus robustus robustus TM Swartkrans, South Africa SK_843 LM1 L Y Y

Paranthropus robustus robustus TM Swartkrans, South Africa SK_1 LM2 L Y Y

Paranthropus robustus robustus TM Swartkrans, South Africa SK_1587B LM2 R Y Y

Paranthropus robustus robustus UW Drimolen, South Africa DNH_60c LM2 R Y Y Y

Paranthropus robustus robustus TM Swartkrans, South Africa SK_6 LM2 R Y Y

Paranthropus robustus robustus TM Swartkrans, South Africa SK_843 LM2 L Y Y

Paranthropus robustus robustus TM Kromdraai, South Africa TM_1600 LM2 L Y

Paranthropus robustus robustus TM Swartkrans, South Africa SK_22 LM3 R Y Y

Paranthropus robustus robustus TM Swartkrans, South Africa SK_75 LM3 R Y Y

Paranthropus robustus robustus TM Swartkrans, South Africa SK_851 LM3 R Y Y

Paranthropus robustus robustus TM Swartkrans, South Africa SK_6 LM3 R Y Y

Paranthropus robustus robustus TM Swartkrans, South Africa SK_841b LM3 L Y Y

Paranthropus robustus robustus TM Swartkrans, South Africa SKX_10642 LM3 L Y Y

Paranthropus robustus robustus TM Swartkrans, South Africa SKX_10643 LM3 R Y

Paranthropus robustus robustus TM Swartkrans, South Africa SKX_5002 LM3 L Y Y

Paranthropus robustus robustus TM Swartkrans, South Africa SKX_5014 LM3 R Y Y

1. Source codes: MPI-EVA, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany; NMNH, National Museum of Natural History, Washington, DC, USA; ZMB, Museum für Naturkunde, Humboldt Universität, Berlin, Germany; SFN, Senckenberg Forschungsinstitut und Naturmuseum, Frankfurt, Germany; MNCN, Museo Nacional de Ciencias Naturales, Madrid, Spain, TM, Transvaal Museum, Pretoria, South Africa; UW, University of Witwatersrand, Johannesburg, South Africa.

2. (*) - Indicates that the specimen has no museum accession number and that the listed accession number is an internal number of the MPI-EVA.

185

Appendix B. This appendix is composed of an example of the Mathematica code used to perform a Procrustes superimposition on the landmark data to create a sample of a homologous landmark set for each specimen. In this case the code pertains to the analysis of the combined first and second molar sample of Pan (Chapter 2). Step 1. Identify appropriate data files

Step 2. Import of landmark data

►Reads in reference surface model representing a generic EDJ surface. ►Identifies the MAIN, RIDGE and CERVIX landmark text files. In this case for both first and second molars of the Pan sample. ►Gets names of specimens from landmark files ►Designates the reference specimen ►Imports the landmark data.

186

Step 3. Create and sample EDJ RIDGE curve

Step 4. Create and sample CERVIX curve

Step 5. Procrustes superimposition of specimens

►Fits a cubic spline function to the MAIN and RIDGE landmarks of each specimen. ►Divides this spline curve into 8 sections based on MAIN landmarks. ►Calculates a number of equally spaced landmarks on the spline curve between adjacent landmarks. For example for “dentin1” it calculates 7 equally spaced landmarks between MAIN landmarks #1 and #6 (see Fig. 2.1) ►Fits a cubic spline function to the CERVIX landmarks of each specimen. ►Calculates 70 equally spaced landmarks along the cubic spline. ►All specimens after Procrustes superimposition.

187

Step 6. Create matrix of Procrustes coordinates

Step 7. Check for outliers

►PCA of Procrustes coordinates to check for outliers due to methodological errors (e.g., mistakes during landmark collection on original EDJ surface). ►Identifies the order of points along each curve

188

Step 8. Slide landmarks on RIDGE and

CERVIX curves

►Illustration of specimen number 5 with landmark locations. ►Calculates vectors between adjacent landmarks that are tangent to the curve and along with landmarks will be allowed to slide. ►Illustration of the vectors calculated for specimen 5. Note vectors are not calculated adjacent to MAIN landmarks because these landmarks are not allowed to slide. ►Writes new matrix of coordinates after sliding.

189

Step 9. Slide landmarks a second time

►Shows displacement of landmarks for specimen #2. ►Calculates Procrustes superimposition on all specimens after first sliding. ►PCA after first sliding. ►Reimpliments the sliding routine

190

►Curves upon which slide landmarks are projected back onto after sliding along their associated vectors. ► Shows displacement of landmarks for specimen #2 after second sliding. Note the marked reduction in the degree of landmark displacement compared to first sliding.

191

Step 10. Final Procrustes superimposition

Step 11. Export of homologous landmark

datasets

►Exports the homologous landmark sets for each specimen. ►Lists centroid sizes for each specimen.